Method of making a device for generating droplets

ABSTRACT

Methods of making a droplet-generating device. In an exemplary method, an upper member is injection molded. The upper member includes a bottom surface and also includes a first microfluidic channel, a second microfluidic channel, and a third microfluidic channel each formed in the bottom surface. The upper member has a plurality of openings each extending completely through the upper member from the bottom surface and creating a side wall region of a sample well, a carrier well, and a droplet well. A cover layer is attached to the bottom surface of the upper member, such that the cover layer seals a bottom side of each microfluidic channel. The microfluidic channels meet one another to create a droplet-generation region. The sample well, the carrier well, and the droplet well are connected to the droplet-generation region via the first, second, and third microfluidic channels, respectively.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/962,511, filed Dec. 7, 2010, which in turn is a continuation of U.S.patent application Ser. No. 12/586,626, filed Sep. 23, 2009, now U.S.Pat. No. 9,156,010, which in turn is based upon and claims the benefitunder 35 U.S.C. §119(e) of the following U.S. provisional patentapplications: Ser. No. 61/194,043, filed Sep. 23, 2008; Ser. No.61/206,975, filed Feb. 5, 2009; Ser. No. 61/271,538, filed Jul. 21,2009; Ser. No. 61/275,731, filed Sep. 1, 2009; Ser. No. 61/277,200,filed Sep. 21, 2009; Ser. No. 61/277,203, filed Sep. 21, 2009; Ser. No.61/277,204, filed Sep. 21, 2009; Ser. No. 61/277,216, filed Sep. 21,2009; Ser. No. 61/277,249, filed Sep. 21, 2009; and Ser. No. 61/277,270,filed Sep. 22, 2009. These priority applications are incorporated hereinby reference in their entireties for all purposes.

CROSS-REFERENCES TO OTHER MATERIALS

This application incorporates by reference in their entireties for allpurposes the following materials: U.S. Pat. No. 7,041,481, issued May 9,2006; and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY(2nd Ed. 1999).

INTRODUCTION

Assays are procedures for determining the presence, quantity, activity,and/or other properties or characteristics of components in a sample. Inmany cases, the samples to be assayed are complex, the components ofinterest within the samples—a nucleic acid, an enzyme, a virus, abacterium, etc.—are only minor constituents of the samples, and theresults of the assays are required quickly and/or for many samples.Unfortunately, current assay systems, such as polymerase chain reaction(PCR) assays for nucleic acids such as deoxyribonucleic acid (DNA), maybe slow, sensitive to sample complexity, and/or prone to reporting falsepositives, among other disadvantages. Thus, there is a need for improvedassay systems.

SUMMARY

The present disclosure provides methods of making a droplet-generatingdevice. In an exemplary method, an upper member is injection molded. Theupper member includes a bottom surface and also includes a firstmicrofluidic channel, a second microfluidic channel, and a thirdmicrofluidic channel each formed in the bottom surface. The upper memberhas a plurality of openings each extending completely through the uppermember from the bottom surface and creating a side wall region of asample well, a carrier well, and a droplet well. A cover layer isattached to the bottom surface of the upper member, such that the coverlayer seals a bottom side of each microfluidic channel. The microfluidicchannels meet one another to create a droplet-generation region. Thesample well, the carrier well, and the droplet well are connected to thedroplet-generation region via the first, second, and third microfluidicchannels, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart listing exemplary steps that may be performed in amethod of sample analysis using droplet-based assays, in accordance withaspects of the present disclosure.

FIG. 2 is a perspective view of an exemplary embodiment of a system forperforming droplet-based assays, with the system comprising aninstrument and cartridges that connect to the instrument to providesample preparation that is actuated and controlled by the instrument, inaccordance with aspects of the present disclosure.

FIG. 3A is a schematic view of an exemplary sequence of processesperformed by the system of FIG. 2.

FIG. 3B is a schematic view of the instrument of FIG. 2.

FIG. 4 is a perspective view of another exemplary embodiment of aninstrument for performing droplet-based assays, with the instrumentdesigned to utilize pre-prepared samples, in accordance with aspects ofthe present disclosure.

FIG. 5 is a flowchart listing exemplary steps that may be performed in amethod of sample analysis using droplet-based assays, in accordance withaspects of the present disclosure.

FIG. 6 is a schematic view of selected portions of an exemplary systemfor performing droplet-based assays, in accordance with aspects of thepresent disclosure.

FIG. 7 is a schematic view of an exemplary system with flow-basedamplification, and with droplet generation and droplet loading that aredecoupled from each other, in accordance with aspects of the presentdisclosure.

FIG. 8 is a flowchart listing exemplary steps that may be performed in amethod of sample analysis using droplet-based assays in which dropletsare transported from a droplet generator and/or a droplet storage siteto a reaction site, in accordance with aspects of the presentdisclosure.

FIG. 9 is a flowchart listing exemplary steps that may be included in adroplet transport step in the method of FIG. 8, in accordance withaspects of the present disclosure.

FIG. 10 is a schematic view of selected portions of an exemplary systemfor performing droplet-based assays in which droplets are transportedfrom a droplet generator and/or droplet storage site to a reaction site,with horizontal arrows indicating droplet travel between structuralcomponents of the system, in accordance with aspects of the presentdisclosure.

FIG. 11 is a schematic view of an exemplary droplet transporterconnecting a droplet storage site to a reaction site, in accordance withaspects of the present disclosure.

FIG. 12 is a schematic view of an example of the system of FIG. 10 inwhich droplet generation and droplet transport to a reaction site arecoupled by continuous flow such that droplets are not stored, inaccordance with aspects of the present disclosure.

FIG. 13 is a schematic view of an example of the system of FIG. 10 inwhich droplet generation and droplet transport to a reaction site aredecoupled, such that droplets can be stored for an adjustable,selectable period of time after their generation and then loaded intothe reaction site for droplet processing, in accordance with aspects ofthe present disclosure.

FIG. 14 is a schematic view of an example of a system generally relatedto the system of FIG. 13, with selected elements replicated such thatthe system is capable of transporting, reacting, and/or detecting aplurality of distinct droplet packets in parallel, in accordance withaspects of the present disclosure.

FIG. 15 is a schematic view of another example of the system of FIG. 10in which droplet generation and droplet transport to a reaction site aredecoupled, with the system utilizing an autosampler to transportselected droplet packets from an emulsion array to a reaction site, inaccordance with aspects of present disclosure.

FIG. 16 is a fragmentary view of selected portions of the system of FIG.15, with the autosampler picking up droplet packets serially from theemulsion array and separated from one another by at least one spacerfluid, in accordance with aspects of present disclosure.

FIG. 17 is a schematic, fragmentary view of an example of the system ofFIG. 10 that enables multi-stage decoupling of droplet generation anddroplet loading into a reaction site, with the system providing storageof a packet of droplets (a) as part of an array of emulsions and then(b) in an intermediate storage site prior to introducing the packet intoa reaction site, in accordance with aspects of the present disclosure.

FIG. 18 is a schematic, fragmentary view of another example of thesystem of FIG. 10 that enables multi-stage decoupling of dropletgeneration and droplet loading into a reaction site, with the systemrelated to that of FIG. 17 but including a plurality of isolated,intermediate storage sites that can be accessed in an arbitrary order,in accordance with aspects of present disclosure.

FIG. 19 is a flowchart listing exemplary steps that may be performed ina method of sample analysis using droplets subjected to conditions foramplification while disposed in a static fluid, in accordance withaspects of present disclosure.

FIG. 20 is a flowchart listing exemplary steps that may be performed ina method of sample analysis using parallel (batch) amplification of anarray of emulsions, in accordance with aspects of the presentdisclosure.

FIG. 21 is a schematic view of selected portions of an exemplary systemfor performing the method of FIG. 20, in accordance with aspects of thepresent disclosure.

FIG. 22 is a view of an exemplary device equipped with an array ofdroplet generators, in accordance with aspects of the presentdisclosure.

FIG. 23 is a fragmentary view of the device of FIG. 22, taken generallyat the region indicated at “23” in FIG. 22, and illustrating a subset ofthe droplet generators.

FIG. 24 is a schematic view of one of the droplet generators of FIG. 23,illustrating how droplets are generated and driven to a dropletreservoir by application of pressure.

FIG. 25 is a sectional view of the device of FIG. 22, taken generallyalong line 25-25 of FIG. 23, and with the device assembled with anexemplary pressure manifold for applying pressure to the dropletgenerators to drive droplet generation, in accordance with aspects ofpresent disclosure.

FIG. 26 is a sectional view of the device of FIG. 22 taken as in FIG.25, but with the pressure manifold replaced by an exemplary sealingmember that seals wells of the device to permit thermal cycling, inaccordance with aspects of present disclosure.

FIG. 27 is a fragmentary view of another exemplary device incorporatingan array of droplet generators, in accordance with aspects of presentdisclosure.

FIG. 28 is a bottom view of a droplet generator of the device of FIG.27, taken after droplet generation.

FIG. 29 is a sectional view of the droplet generator of FIG. 28, takengenerally along line 29-29 of FIG. 28 and illustrating how droplets maybe imaged from below the device.

FIG. 30 is a fragmentary view of yet another exemplary deviceincorporating an array of droplet generators, in accordance with aspectsof present disclosure.

FIG. 31 is a bottom view of a droplet generator of the device of FIG.30, taken after droplet generation.

FIG. 32 is a sectional view of the droplet generator of FIG. 31, takengenerally along line 32-32 of FIG. 31 and illustrating how droplets maybe imaged from below the device.

FIG. 33 is a view of an exemplary imaging system for batch detection ofan array of emulsions held by a plate, in accordance with aspects of thepresent disclosure.

FIG. 34 is a sectional view of the plate of FIG. 33, taken through awell of the plate, generally along line 34-34 of FIG. 33.

FIG. 35 is a view of an exemplary imaging system for detecting images ofemulsions held by slides, in accordance with aspects of the presentdisclosure.

FIG. 36 is a sectional view through a slide of the imaging system ofFIG. 35, taken generally along line 36-36 of FIG. 35.

FIG. 37 is an exploded view of an exemplary imaging system that includesa vial being loaded with droplets before detection to image thedroplets, in accordance with aspects of present disclosure.

FIG. 38 is a schematic view of an exemplary system for imaging amplifiedemulsions by transport of droplets of the emulsions to a detectionchamber by flow from a plate holding the emulsions, in accordance withaspects of the present disclosure.

FIG. 39 is a schematic view of an exemplary system for imaging amplifiedemulsions transported to a plurality of detection chambers by flow froma plate holding the emulsions, in accordance with aspects of the presentdisclosure.

FIG. 40 is a schematic view of an exemplary system for transport ofdroplets from an array of emulsions to a detection channel, inaccordance aspects of the present disclosure.

FIG. 41 is a flowchart depicting the steps of a DNA amplification methodthat may be performed within or in conjunction with a disposablecartridge of a DNA amplification system, in accordance with aspects ofthe present disclosure.

FIG. 42 is a schematic diagram depicting a disposable sample preparationcartridge and suitable fluidic connections between various components ofthe cartridge, in accordance with aspects of the present disclosure.

FIGS. 43-45 are isometric, side elevation, and top views, respectively,of an interior portion of an exemplary disposable cartridge, suitablefor performing some or all of the sample preparation steps in FIG. 41.

FIG. 46 is a schematic view of a two-chamber hydraulic mechanism,suitable for controlling fluid motion between the various chambers of adisposable cartridge, in accordance with aspects of the presentdisclosure.

FIG. 47 is a schematic view of a three-chamber hydraulic mechanism,which is similar to two-chamber mechanism of FIG. 46, suitable forcontrolling fluid motion between the various chambers of a disposablecartridge, in accordance with aspects of the present disclosure.

FIGS. 48A-48F are top views of various exemplary droplet generators, inaccordance with aspects of the present disclosure.

FIG. 49 is a schematic diagram depicting another disposable samplepreparation cartridge and suitable fluidic connections between variouscomponents of the cartridge, in accordance with aspects of the presentdisclosure.

FIG. 50 is a schematic diagram depicting still another disposable samplepreparation cartridge (left), portions of a complementary PCR instrument(right), and suitable fluidic connections among and between variouscomponents of the cartridge and instrument, in accordance with aspectsof the present disclosure.

FIG. 51 is a schematic diagram depicting still another disposable samplepreparation cartridge (left), portions of a complementary PCR instrument(right), and suitable fluidic connections among and between variouscomponents of the cartridge and instrument, in accordance with aspectsof the present disclosure.

FIG. 52 is an isometric view of still another disposable samplepreparation cartridge, in accordance with aspects of the presentdisclosure.

FIG. 53 is a bottom view of the cartridge of FIG. 52.

FIG. 54 is a schematic diagram of an exemplary droplet generationsystem, in accordance with aspects of the present disclosure.

FIG. 55 is an isometric view of a portion of an exemplary dropletgenerator, in accordance with aspects of the present disclosure.

FIG. 56 is an isometric view of a portion of another exemplary dropletgenerator, in accordance with aspects of the present disclosure.

FIG. 57 is a cross-sectional side elevational view showing an innerportion of another exemplary droplet generator, in accordance withaspects of the present disclosure.

FIG. 58 is a cross-sectional side elevational view showing an innerportion of another exemplary droplet generator, in accordance withaspects of the present disclosure.

FIG. 59 is a cross-sectional side elevational view showing an innerportion of another exemplary droplet generator, in accordance withaspects of the present disclosure, showing a sample-containing portiondisassembled from a droplet outlet portion.

FIG. 60 is a cross-sectional side elevational view showing thesample-containing portion and the droplet outlet portion of FIG. 59assembled together.

FIG. 61 is a cross-sectional side elevational view of a dropletgeneration system including a droplet generator and a fluid reservoir,in accordance with aspects of the present disclosure.

FIG. 62 is a magnified cross-sectional side elevational view of a distalportion of the droplet generation system of FIG. 61.

FIG. 63 is a cross-sectional side elevational view of a distal portionof another droplet generation system, in accordance with aspects of thepresent disclosure.

FIG. 64 is a cross-sectional side elevational view of a distal portionof yet another droplet generation system, in accordance with aspects ofthe present disclosure.

FIG. 65 is a cross-sectional side elevational view of still anotherdroplet generation system, in accordance with aspects of the presentdisclosure.

FIG. 66 is a cross-sectional side elevational view of still anotherdroplet generation system, in accordance with aspects of the presentdisclosure.

FIG. 67 is a cross-sectional side elevational view of still anotherdroplet generation system, in accordance with aspects of the presentdisclosure.

FIG. 68 is a cross-sectional side elevational view of still anotherdroplet generation system, in accordance with aspects of the presentdisclosure.

FIG. 69 is an isometric view of four different droplet generators,illustrating the relationship between various cross-type dropletgenerators, in accordance with aspects of the present disclosure

FIG. 70 is a cross-sectional side elevational view of another dropletgeneration system, in accordance with aspects of the present disclosure.

FIG. 71 is a cross-sectional side elevational view of still anotherdroplet generation system, in accordance with aspects of the presentdisclosure.

FIG. 72 is a flowchart depicting a method of thermocycling asample/reagent fluid mixture to promote PCR.

FIG. 73 is an exploded isometric view of an exemplary thermocycler, inaccordance with aspects of the present disclosure.

FIG. 74 is an unexploded isometric view of a central portion of thethermocycler of FIG. 73.

FIG. 75 is an isometric view showing a magnified portion of theassembled thermocycler of FIG. 73, which is suitable for relativelysmall outer diameter fluidic tubing, in accordance with aspects of thepresent disclosure.

FIG. 76 is an isometric view showing a magnified portion of analternative embodiment of the assembled thermocycler, which is suitablefor relatively larger outer diameter fluidic tubing, in accordance withaspects of the present disclosure.

FIG. 77 is a top plan view of the thermocycler of FIG. 73, without theouter segments attached.

FIG. 78 is a schematic sectional view of the thermocycler of FIG. 73,depicting the relative dispositions of the core and other components,taken generally along line C in FIG. 77 as line C in swept through oneclockwise revolution about the center of the thermocycler.

FIG. 79 is a magnified isometric view of a central portion of thethermocycler of FIG. 75.

FIG. 80 is a graph of measured temperature versus arc length, as afunction of average fluid velocity, near the interface between two innersegments of the thermocycler of FIG. 73.

FIG. 81 is an isometric view of a central portion of a thermocyclerhaving an optional “hot start” region, in accordance with aspects of thepresent disclosure.

FIGS. 82-89 are schematic sectional views of alternative embodiments ofa thermocycler, in accordance with aspects of the present disclosure.

FIG. 90 is an exploded isometric view of a thermocycler, with associatedheating, cooling, and housing elements, in accordance with aspects ofthe present disclosure.

FIG. 91 is a side elevational view of an exemplary thermocycler havingtemperature regions that vary in size along the length of thethermocycler, in accordance with aspects of the present disclosure.

FIG. 92 is a side elevational view of an exemplary thermocycler havingtemperature regions that vary in number along the length of thethermocycler, in accordance with aspects of the present disclosure.

FIG. 93 is a schematic depiction of an optical detection system forirradiating sample-containing droplets and detecting fluorescencesubsequently emitted by the droplets, in accordance with aspects of thepresent disclosure.

FIG. 94 is a graph of intensity versus time for fluorescence detected byan optical detection system such as the system of FIG. 93, illustratingthe distinction between fluorescence emitted by droplets containing atarget and droplets not containing a target.

FIG. 95 is a schematic depiction of an optical detection system in whichstimulating radiation is transferred toward sample-containing dropletsthrough an optical fiber, in accordance with aspects of the presentdisclosure.

FIG. 96 is a schematic depiction of an optical detection system in whichscattered and fluorescence radiation are transferred away fromsample-containing droplets through optical fibers, in accordance withaspects of the present disclosure.

FIG. 97 is a schematic depiction of an optical detection system in whichstimulating radiation is transferred toward sample-containing dropletsthrough an optical fiber and in which scattered and fluorescenceradiation are transferred away from the droplets through optical fibers,in accordance with aspects of the present disclosure.

FIG. 98 depicts an intersection region where incident radiationintersects with sample-containing droplets traveling through a fluidchannel, illustrating how optical fibers may be integrated with sectionsof fluidic tubing.

FIG. 99A depicts another intersection region where incident radiationintersects with sample-containing droplets traveling through a fluidchannel, illustrating how a single optical fiber may be used to transmitboth incident radiation and stimulated fluorescence.

FIG. 99B depicts another intersection region configured to transmit bothincident radiation and stimulated fluorescence through a single opticalfiber, and also configured to transfer radiation to and fromsubstantially one droplet at a time.

FIG. 100 is a schematic depiction of an optical detection system inwhich the incident radiation is split into a plurality of separatebeams, in accordance with aspects of the present disclosure.

FIG. 101 is a schematic depiction of an optical detection system inwhich the incident radiation is spread by an adjustable mirror into arelatively wide intersection region, in accordance with aspects of thepresent disclosure.

FIG. 102 depicts a flow focus mechanism for separating sample-containingdroplets from each other by a desired distance, in accordance withaspects of the present disclosure.

FIG. 103 depicts another flow focus mechanism for separatingsample-containing droplets from each other by a desired distance, inaccordance with aspects of the present disclosure.

FIG. 104 depicts a section of fluidic tubing, illustrating how anappropriate choice of fluid channel diameter can facilitate properspacing between droplets, in accordance with aspects of the presentdisclosure.

FIG. 105 depicts a batch fluorescence detection system, in accordancewith aspects of the present disclosure.

FIG. 106 is a flow chart depicting a method of detecting fluorescencefrom sample-containing droplets, in accordance with aspects of thepresent disclosure.

FIG. 107 is a flowchart depicting a method of determining targetmolecule concentration in a plurality of sample-containing droplets, inaccordance with aspects of the present disclosure.

FIG. 108 is a histogram showing exemplary experimental data in which thenumber of detected droplets is plotted as a function of a measure offluorescence intensity.

FIG. 109 is a histogram comparing the experimental data in FIG. 108(solid line) with fluorescence distributions recreated numerically usingvarious fit orders (dotted and dashed lines).

FIG. 110 is a histogram showing values of least mean square residualsfor the fluorescence distributions of FIG. 108 recreated numericallyusing various fit orders.

FIG. 111 is a flowchart depicting a method of numerically estimatingtarget molecule concentration in a sample, in accordance with aspects ofthe present disclosure.

FIG. 112 is an exemplary graph of fluorescence signals that may bemeasured with respect to time from a flow stream of droplets, with thegraph exhibiting a series of peaks representing droplet signals, andwith the graph indicating a signal threshold for assigning dropletsignals as corresponding to amplification-positive andamplification-negative droplets, in accordance with aspects of thepresent disclosure.

FIG. 113 is an exemplary histogram of ranges of droplet signalintensities that may be measured from the flow stream of FIG. 112, withthe relative frequency of occurrence of each range indicated by barheight, in accordance with aspects of the present disclosure.

FIG. 114 is a schematic view of an exemplary system for performingdroplet-based tests of nucleic acid amplification with the aid ofcontrols and/or calibrators, in accordance with aspects of the presentdisclosure.

FIG. 115 is a schematic view of selected aspects of the system of FIG.114, with the system in an exemplary configuration for detectingamplification of a nucleic acid target using a first dye, and forcontrolling for system variation during a test using a second dye, inaccordance with aspects of present disclosure.

FIG. 116 is a schematic view of exemplary reagents that may be includedin the system configuration of FIG. 115, to permit detection ofamplification signals in a first detector channel and detection of apassive control signals in a second detector channel, in accordance withaspects of present disclosure.

FIG. 117 a flowchart of an exemplary approach to correcting for systemvariation using the system configuration of FIG. 115, in accordance withaspects of the present disclosure.

FIG. 118 is a schematic view of selected aspects of the system of FIG.114, with the system in an exemplary configuration for detectingamplification of a nucleic acid target using a first dye in a set ofdroplets, and for (a) calibrating the system before, during, and/orafter a test or (b) controlling for aspects of system variation during atest using either the first dye or a second dye in another set ofdroplets, in accordance with aspects of present disclosure.

FIG. 119 is an exemplary graph of fluorescence signals that may bedetected over time from a flow stream of the system configuration ofFIG. 118 during system calibration and sample testing performedserially, in accordance with aspects of present disclosure.

FIG. 120 is a flowchart of an exemplary method of correcting for systemvariation produced during a test using the system configuration of FIG.118, in accordance with aspects of the present disclosure.

FIG. 121 is a schematic view of selected aspects of the system of FIG.114, with the system in an exemplary configuration for testingamplification of a pair of nucleic acid targets in the same droplets, inaccordance with aspects of present disclosure.

FIG. 122 is a schematic view of selected aspects of the system of FIG.114, with the system in another exemplary configuration for testingamplification of a pair of nucleic acid targets in the same droplets, inaccordance with aspects of present disclosure.

FIG. 123 is a schematic view of exemplary target-specific reagents thatmay be included in the system configurations of FIGS. 121 and 122, topermit detection of amplification signals in a different detectorchannel (i.e., a different detected wavelength or wavelength range) foreach nucleic acid target, in accordance with aspects of presentdisclosure.

FIG. 124 is a pair of exemplary graphs of fluorescence signals that maybe detected over time from a flow stream of the system configuration ofFIG. 121 or 122 using different detector channels, with one of thechannels detecting successful amplification of a control target, therebyindicating no inhibition of amplification, in accordance with aspects ofpresent disclosure.

FIG. 125 is a pair of exemplary graphs with fluorescence signalsdetected generally as in FIG. 124, but with control signals indicatingthat amplification is inhibited, in accordance with aspects of presentdisclosure.

FIG. 126 is a schematic view of selected aspects of the system of FIG.114, with the system in an exemplary configuration for testingamplification of a pair of nucleic acid targets using a different set ofdroplets for each target, in accordance with aspects of presentdisclosure.

FIG. 127 is a pair of exemplary graphs of fluorescence signals that maybe detected over time from a flow stream of the system configuration ofFIG. 126 using different detector channels, with each channel monitoringamplification of a distinct nucleic acid target, in accordance withaspects of present disclosure.

FIG. 128 is a pair of graphs illustrating exemplary absorption andemission spectra of fluorescent dyes that may be suitable for use in thesystem of FIG. 114, in accordance with aspects of the presentdisclosure.

FIG. 129 is a schematic diagram illustrating exemplary use of thefluorescent dyes of FIG. 128 in an exemplary embodiment of the system ofFIG. 114, in accordance with aspects of the present disclosure.

FIG. 130 is a flowchart of an exemplary approach to correcting forsystem variation within a test by processing a set of droplet testsignals to a more uniform signal intensity, in accordance with aspectsof the present disclosure.

FIG. 131 is a flowchart of an exemplary approach for transformingdroplet signals based on the width of respective signal peaks providingthe droplet signals, in accordance with aspects of the presentdisclosure.

DETAILED DESCRIPTION

The present disclosure provides systems, including apparatus andmethods, for performing assays. These systems may involve, among others,(A) preparing a sample, such as a clinical or environmental sample, foranalysis, (B) separating components of the samples by partitioning theminto droplets or other partitions, each containing only about onecomponent (such as a single copy of a nucleic acid target (DNA or RNA)or other analyte of interest), (C) amplifying or otherwise reacting thecomponents within the droplets, (D) detecting the amplified or reactedcomponents, or characteristics thereof, and/or (E) analyzing theresulting data. In this way, complex samples may be converted into aplurality of simpler, more easily analyzed samples, with concomitantreductions in background and assay times.

FIG. 1 shows an exemplary system 500 for performing such a droplet-, orpartition-, based assay. In brief, the system may include samplepreparation 502, droplet generation 504, reaction (e.g., amplification)506, detection 508, and data analysis 510. The system may be utilized toperform a digital PCR (polymerase chain reaction) analysis. Morespecifically, sample preparation 502 may involve collecting a sample,such as a clinical or environmental sample, treating the sample torelease associated nucleic acids, and forming a reaction mixtureinvolving the nucleic acids (e.g., for amplification of a target nucleicacid). Droplet generation 504 may involve encapsulating the nucleicacids in droplets, for example, with about one copy of each targetnucleic acid per droplet, where the droplets are suspended in animmiscible carrier fluid, such as oil, to form an emulsion. Reaction 506may involve subjecting the droplets to a suitable reaction, such asthermal cycling to induce PCR amplification, so that target nucleicacids, if any, within the droplets are amplified to form additionalcopies. Detection 508 may involve detecting some signal(s) from thedroplets indicative of whether or not there was amplification. Finally,data analysis 510 may involve estimating a concentration of the targetnucleic acid in the sample based on the percentage of droplets in whichamplification occurred.

These and other aspects of the system are described below, in thefollowing sections: (I) definitions, (II) system overview/architecture,(III) sample preparation/cartridge, (IV) droplet generator, (V)continuous flow thermocycler, (VI) detection, (VII)quantification/analysis, (VIII) controls and calibrations, (IX) clinicalapplications, and (X) multiplexed assays.

I. DEFINITIONS

Technical terms used in this disclosure have the meanings that arecommonly recognized by those skilled in the art. However, the followingterms may have additional meanings, as described below.

Emulsion—a composition comprising liquid droplets disposed in animmiscible carrier fluid, which also is liquid. The carrier fluid, alsotermed a background fluid, forms a continuous phase, which may be termeda carrier phase, a carrier, and/or a background phase. The droplets(e.g., aqueous droplets) are formed by at least one droplet fluid, alsotermed a foreground fluid, which is a liquid and which forms a dropletphase (which may be termed a dispersed phase or discontinuous phase).The droplet phase is immiscible with the continuous phase, which meansthat the droplet phase (i.e., the droplets) and the continuous phase(i.e., the carrier fluid) do not mix to attain homogeneity. The dropletsare isolated from one another by the continuous phase and encapsulated(i.e., enclosed/surrounded) by the continuous phase.

The droplets of an emulsion may have any uniform or non-uniformdistribution in the continuous phase. If non-uniform, the concentrationof the droplets may vary to provide one or more regions of higherdroplet density and one or more regions of lower droplet density in thecontinuous phase. For example, droplets may sink or float in thecontinuous phase, may be clustered in one or more packets along achannel, may be focused toward the center or perimeter of a flow stream,or the like.

Any of the emulsions disclosed herein may be monodisperse, that is,composed of droplets of at least generally uniform size, or may bepolydisperse, that is, composed of droplets of various sizes. Ifmonodisperse, the droplets of the emulsion may, for example, vary involume by a standard deviation that is less than about plus or minus100%, 50%, 20%, 10%, 5%, 2% or 1% of the average droplet volume.Droplets generated from an orifice may be monodisperse or polydisperse.

An emulsion may have any suitable composition. The emulsion may becharacterized by the predominant liquid compound or type of liquidcompound in each phase. The predominant liquid compounds in the emulsionmay be water and oil. “Oil” is any liquid compound or mixture of liquidcompounds that is immiscible with water and that has a high content ofcarbon. In some examples, oil also may have a high content of hydrogen,fluorine, silicon, oxygen, or any combination thereof, among others. Forexample, any of the emulsions disclosed herein may be a water-in-oil(W/O) emulsion (i.e., aqueous droplets in a continuous oil phase). Theoil may, for example, be or include at least one silicone oil, mineraloil, fluorocarbon oil, vegetable oil, or a combination thereof, amongothers. Any other suitable components may be present in any of theemulsion phases, such as at least one surfactant, reagent, sample (i.e.,partitions thereof), other additive, label, particles, or anycombination thereof.

Standard emulsions become unstable when heated (e.g., to temperaturesabove 60° C.) when they are in a packed state (e.g., each droplet isnear a neighboring droplet), because heat generally lowers interfacialtensions, which can lead to droplet coalescence. Thus, standard packedemulsions do not maintain their integrity during high-temperaturereactions, such as PCR, unless emulsion droplets are kept out of contactwith one another or additives (e.g., other oil bases, surfactants, etc.)are used to modify the stability conditions (e.g., interfacial tension,viscosity, steric hindrance, etc.). For example, the droplets may bearranged in single file and spaced from one another along a channel topermit thermal cycling in order to perform PCR. However, following thisapproach using a standard emulsion does not permit a high density ofdroplets, thereby substantially limiting throughput in droplet-basedassays.

Any emulsion disclosed herein may be a heat-stable emulsion. Aheat-stable emulsion is any emulsion that resists coalescence whenheated to at least 50° C. A heat-stable emulsion may be a PCR-stableemulsion, which is an emulsion that resists coalescence throughout thethermal cycling of PCR (e.g., to permit performance of digital PCR).Accordingly, a PCR-stable emulsion may be resistant to coalescence whenheated to at least 80° C. or 90° C., among others. Due to heatstability, a PCR-stable emulsion, in contrast to a standard emulsion,enables PCR assays to be performed in droplets that remain substantiallymonodisperse throughout thermal cycling. Accordingly, digital PCR assayswith PCR-stable emulsions may be substantially more quantitative thanwith standard emulsions. An emulsion may be formulated as PCR stable by,for example, proper selection of carrier fluid and surfactants, amongothers. An exemplary oil formulation to generate PCR-stable emulsionsfor flow-through assays is as follows: (1) Dow Corning 5225C FormulationAid (10% active ingredient in decamethylcyclopentasiloxane)—20% w/w, 2%w/w final concentration active ingredient, (2) Dow Corning 749 Fluid(50% active ingredient in decamethylcyclopentasiloxane)—5% w/w, 2.5% w/wactive ingredient, and (3) Poly(dimethylsiloxane) Dow Corning 200®fluid, viscosity 5.0 cSt (25° C.)—75% w/w. An exemplary oil formulationto generate PCR-stable emulsions for batch assays is as follows: (1) DowCorning 5225C Formulation Aid (10% active ingredient indecamethylcyclopentasiloxane)—20% w/w, 2% w/w final concentration activeingredient, (2) Dow Corning 749 Fluid (50% active ingredient indecamethylcyclopentasiloxane)—60% w/w, 30% w/w active ingredient, and(3) Poly(dimethylsiloxane) Dow Corning 200® fluid, viscosity 5.0 cSt(25° C.)—20% w/w.

Partition—a separated portion of a bulk volume. The partition may be asample partition generated from a sample, such as a prepared sample,that forms the bulk volume. Partitions generated from a bulk volume maybe substantially uniform in size or may have distinct sizes (e.g., setsof partitions of two or more discrete, uniform sizes). Exemplarypartitions are droplets. Partitions may also vary continuously in sizewith a predetermined size distribution or with a random sizedistribution.

Droplet—a small volume of liquid, typically with a spherical shape,encapsulated by an immiscible fluid, such as a continuous phase of anemulsion. The volume of a droplet, and/or the average volume of dropletsin an emulsion, may, for example, be less than about one microliter(i.e., a “microdroplet”) (or between about one microliter and onenanoliter or between about one microliter and one picoliter), less thanabout one nanoliter (or between about one nanoliter and one picoliter),or less than about one picoliter (or between about one picoliter and onefemtoliter), among others. A droplet (or droplets of an emulsion) mayhave a diameter (or an average diameter) of less than about 1000, 100,or 10 micrometers, or of about 1000 to 10 micrometers, among others. Adroplet may be spherical or nonspherical. A droplet may be a simpledroplet or a compound droplet, that is, a droplet in which at least onedroplet encapsulates at least one other droplet.

Surfactant—a surface-active agent capable of reducing the surfacetension of a liquid in which it is dissolved, and/or the interfacialtension with another phase. A surfactant, which also or alternativelymay be described as a detergent and/or a wetting agent, incorporatesboth a hydrophilic portion and a hydrophobic portion, which collectivelyconfer a dual hydrophilic-lipophilic character on the surfactant. Asurfactant may be characterized according to a Hydrophile-LipophileBalance (HLB) value, which is a measure of the surfactant'shydrophilicity compared to its lipophilicity. HLB values range from 0-60and define the relative affinity of a surfactant for water and oil.Nonionic surfactants generally have HLB values ranging from 0-20 andionic surfactants may have HLB values of up to 60. Hydrophilicsurfactants have HLB values greater than about 10 and a greater affinityfor water than oil. Lipophilic surfactants have HLB values less thanabout 10 and a greater affinity for oil than water. The emulsionsdisclosed herein and/or any phase thereof, may include at least onehydrophilic surfactant, at least one lipophilic surfactant, or acombination thereof. Alternatively, or in addition, the emulsionsdisclosed herein and/or any phase thereof, may include at least onenonionic (and/or ionic) detergent. Furthermore, an emulsion disclosedherein and/or any phase thereof may include a surfactant comprisingpolyethyleneglycol, polypropyleneglycol, or Tween 20, among others.

Packet—a set of droplets or other isolated partitions disposed in thesame continuous volume or volume region of a continuous phase. A packetthus may, for example, constitute all of the droplets of an emulsion ormay constitute a segregated fraction of such droplets at a positionalong a channel. Typically, a packet refers to a collection of dropletsthat when analyzed in partial or total give a statistically relevantsampling to quantitatively make a prediction regarding a property of theentire starting sample from which the initial packet of droplets wasmade. The packet of droplets also indicates a spatial proximity betweenthe first and the last droplets of the packet in a channel.

As an analogy with information technology, each droplet serves as a“bit” of information that may contain sequence specific information froma target analyte within a starting sample. A packet of droplets is thenthe sum of all these “bits” of information that together providestatistically relevant information on the analyte of interest from thestarting sample. As with a binary computer, a packet of droplets isanalogous to the contiguous sequence of bits that comprises the smallestunit of binary data on which meaningful computations can be applied. Apacket of droplets can be encoded temporally and/or spatially relativeto other packets that are also disposed in a continuous phase (such asin a flow stream), and/or with the addition of other encoded information(optical, magnetic, etc.) that uniquely identifies the packet relativeto other packets.

Test—a procedure(s) and/or reaction(s) used to characterize a sample,and any signal(s), value(s), data, and/or result(s) obtained from theprocedure(s) and/or reaction(s). A test also may be described as anassay. Exemplary droplet-based assays are biochemical assays usingaqueous assay mixtures. More particularly, the droplet-based assays maybe enzyme assays and/or binding assays, among others. The enzyme assaysmay, for example, determine whether individual droplets contain a copyof a substrate molecule (e.g., a nucleic acid target) for an enzymeand/or a copy of an enzyme molecule. Based on these assay results, aconcentration and/or copy number of the substrate and/or the enzyme in asample may be estimated.

Reaction—a chemical reaction, a binding interaction, a phenotypicchange, or a combination thereof, which generally provides a detectablesignal (e.g., a fluorescence signal) indicating occurrence and/or anextent of occurrence of the reaction. An exemplary reaction is an enzymereaction that involves an enzyme-catalyzed conversion of a substrate toa product.

Any suitable enzyme reactions may be performed in the droplet-basedassays disclosed herein. For example, the reactions may be catalyzed bya kinase, nuclease, nucleotide cyclase, nucleotide ligase, nucleotidephosphodiesterase, polymerase (DNA or RNA), prenyl transferase,pyrophospatase, reporter enzyme (e.g., alkaline phosphatase,beta-galactosidase, chloramphenicol acetyl transferse, glucuronidase,horse radish peroxidase, luciferase, etc.), reverse transcriptase,topoisomerase, etc.

Sample—a compound, composition, and/or mixture of interest, from anysuitable source(s). A sample is the general subject of interest for atest that analyzes an aspect of the sample, such as an aspect related toat least one analyte that may be present in the sample. Samples may beanalyzed in their natural state, as collected, and/or in an alteredstate, for example, following storage, preservation, extraction, lysis,dilution, concentration, purification, filtration, mixing with one ormore reagents, pre-amplification (e.g., to achieve target enrichment byperforming limited cycles (e.g., <15) of PCR on sample prior to PCR),removal of amplicon (e.g., treatment with uracil-d-glycosylase (UDG)prior to PCR to eliminate any carry-over contamination by a previouslygenerated amplicon (i.e., the amplicon is digestable with UDG because itis generated with dUTP instead of dTTP)), partitioning, or anycombination thereof, among others. Clinical samples may includenasopharyngeal wash, blood, plasma, cell-free plasma, buffy coat,saliva, urine, stool, sputum, mucous, wound swab, tissue biopsy, milk, afluid aspirate, a swab (e.g., a nasopharyngeal swab), and/or tissue,among others. Environmental samples may include water, soil, aerosol,and/or air, among others. Research samples may include cultured cells,primary cells, bacteria, spores, viruses, small organisms, any of theclinical samples listed above, or the like. Additional samples mayinclude foodstuffs, weapons components, biodefense samples to be testedfor bio-threat agents, suspected contaminants, and so on.

Samples may be collected for diagnostic purposes (e.g., the quantitativemeasurement of a clinical analyte such as an infectious agent) or formonitoring purposes (e.g., to determine that an environmental analyte ofinterest such as a bio-threat agent has exceeded a predeterminedthreshold).

Analyte—a component(s) or potential component(s) of a sample that isanalyzed in a test. An analyte is a specific subject of interest in atest where the sample is the general subject of interest. An analytemay, for example, be a nucleic acid, protein, peptide, enzyme, cell,bacteria, spore, virus, organelle, macromolecular assembly, drugcandidate, lipid, carbohydrate, metabolite, or any combination thereof,among others. An analyte may be tested for its presence, activity,and/or other characteristic in a sample and/or in partitions thereof.The presence of an analyte may relate to an absolute or relative number,concentration, binary assessment (e.g., present or absent), or the like,of the analyte in a sample or in one or more partitions thereof. In someexamples, a sample may be partitioned such that a copy of the analyte isnot present in all of the partitions, such as being present in thepartitions at an average concentration of about 0.0001 to 10,000, 0.001to 1000, 0.01 to 100, 0.1 to 10, or one copy per partition.

Reagent—a compound, set of compounds, and/or composition that iscombined with a sample in order to perform a particular test(s) on thesample. A reagent may be a target-specific reagent, which is any reagentcomposition that confers specificity for detection of a particulartarget(s) or analyte(s) in a test. A reagent optionally may include achemical reactant and/or a binding partner for the test. A reagent may,for example, include at least one nucleic acid, protein (e.g., anenzyme), cell, virus, organelle, macromolecular assembly, potentialdrug, lipid, carbohydrate, inorganic substance, or any combinationthereof, and may be an aqueous composition, among others. In exemplaryembodiments, the reagent may be an amplification reagent, which mayinclude at least one primer or at least one pair of primers foramplification of a nucleic acid target, at least one probe and/or dye toenable detection of amplification, a polymerase, nucleotides (dNTPsand/or NTPs), divalent magnesium ions, potassium chloride, buffer, orany combination thereof, among others.

Nucleic acid—a compound comprising a chain of nucleotide monomers. Anucleic acid may be single-stranded or double-stranded (i.e.,base-paired with another nucleic acid), among others. The chain of anucleic acid may be composed of any suitable number of monomers, such asat least about ten or one-hundred, among others. Generally, the lengthof a nucleic acid chain corresponds to its source, with syntheticnucleic acids (e.g., primers and probes) typically being shorter, andbiologically/enzymatically generated nucleic acids (e.g., nucleic acidanalytes) typically being longer.

A nucleic acid may have a natural or artificial structure, or acombination thereof. Nucleic acids with a natural structure, namely,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), generally have abackbone of alternating pentose sugar groups and phosphate groups. Eachpentose group is linked to a nucleobase (e.g., a purine (such as adenine(A) or guanine (T)) or a pyrimidine (such as cytosine (C), thymine (T),or uracil (U))). Nucleic acids with an artificial structure are analogsof natural nucleic acids and may, for example, be created by changes tothe pentose and/or phosphate groups of the natural backbone. Exemplaryartificial nucleic acids include glycol nucleic acids (GNA), peptidenucleic acids (PNA), locked nucleic acid (LNA), threose nucleic acids(TNA), and the like.

The sequence of a nucleic acid is defined by the order in whichnucleobases are arranged along the backbone. This sequence generallydetermines the ability of the nucleic acid to bind specifically to apartner chain (or to form an intramolecular duplex) by hydrogen bonding.In particular, adenine pairs with thymine (or uracil) and guanine pairswith cytosine. A nucleic acid that can bind to another nucleic acid inan antiparallel fashion by forming a consecutive string of such basepairs with the other nucleic acid is termed “complementary.”

Replication—a process forming a copy (i.e., a direct copy and/or acomplementary copy) of a nucleic acid or a segment thereof. Replicationgenerally involves an enzyme, such as a polymerase and/or a ligase,among others. The nucleic acid and/or segment replicated is a template(and/or a target) for replication.

Amplification—a reaction in which replication occurs repeatedly overtime to form multiple copies of at least one segment of a templatemolecule. Amplification may generate an exponential or linear increasein the number of copies as amplification proceeds. Typicalamplifications produce a greater than 1,000-fold increase in copy numberand/or signal. Exemplary amplification reactions for the droplet-basedassays disclosed herein may include the polymerase chain reaction (PCR)or ligase chain reaction, each of which is driven by thermal cycling.The droplet-based assays also or alternatively may use otheramplification reactions, which may be performed isothermally, such asbranched-probe DNA assays, cascade-RCA, helicase-dependentamplification, loop-mediated isothermal amplification (LAMP), nucleicacid based amplification (NASBA), nicking enzyme amplification reaction(NEAR), PAN-AC, Q-beta replicase amplification, rolling circlereplication (RCA), self-sustaining sequence replication,strand-displacement amplification, and the like. Amplification mayutilize a linear or circular template.

Amplification may be performed with any suitable reagents. Amplificationmay be performed, or tested for its occurrence, in an amplificationmixture, which is any composition capable of generating multiple copiesof a nucleic acid target molecule, if present, in the composition. Anamplification mixture may include any combination of at least one primeror primer pair, at least one probe, at least one replication enzyme(e.g., at least one polymerase, such as at least one DNA and/or RNApolymerase), and deoxynucleotide (and/or nucleotide) triphosphates(dNTPs and/or NTPs), among others. Further aspects of assay mixtures anddetection strategies that enable multiplexed amplification and detectionof two or more target species in the same droplet are describedelsewhere herein, such as in Section X, among others.

PCR—nucleic acid amplification that relies on alternating cycles ofheating and cooling (i.e., thermal cycling) to achieve successive roundsof replication. PCR may be performed by thermal cycling between two ormore temperature set points, such as a higher melting (denaturation)temperature and a lower annealing/extension temperature, or among threeor more temperature set points, such as a higher melting temperature, alower annealing temperature, and an intermediate extension temperature,among others. PCR may be performed with a thermostable polymerase, suchas Taq DNA polymerase (e.g., wild-type enzyme, a Stoffel fragment,FastStart polymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tthpolymerase, Vent polymerase, or a combination thereof, among others. PCRgenerally produces an exponential increase in the amount of a productamplicon over successive cycles.

Any suitable PCR methodology or combination of methodologies may beutilized in the droplet-based assays disclosed herein, such asallele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, endpointPCR, hot-start PCR, in situ PCR, intersequence-specific PCR, inversePCR, linear after exponential PCR, ligation-mediated PCR,methylation-specific PCR, miniprimer PCR, multiplex ligation-dependentprobe amplification, multiplex PCR, nested PCR, overlap-extension PCR,polymerase cycling assembly, qualitative PCR, quantitative PCR,real-time PCR, RT-PCR, single-cell PCR, solid-phase PCR, thermalasymmetric interlaced PCR, touchdown PCR, or universal fast walking PCR,among others.

Digital PCR—PCR performed on portions of a sample to determine thepresence/absence, concentration, and/or copy number of a nucleic acidtarget in the sample, based on how many of the sample portions supportamplification of the target. Digital PCR may (or may not) be performedas endpoint PCR. Digital PCR may (or may not) be performed as real-timePCR for each of the partitions.

PCR theoretically results in an exponential amplification of a nucleicacid sequence (analyte) from a sample. By measuring the number ofamplification cycles required to achieve a threshold level ofamplification (as in real-time PCR), one can theoretically calculate thestarting concentration of nucleic acid. In practice, however, there aremany factors that make the PCR process non-exponential, such as varyingamplification efficiencies, low copy numbers of starting nucleic acid,and competition with background contaminant nucleic acid. Digital PCR isgenerally insensitive to these factors, since it does not rely on theassumption that the PCR process is exponential. In digital PCR,individual nucleic acid molecules are separated from the initial sampleinto partitions, then amplified to detectable levels. Each partitionthen provides digital information on the presence or absence of eachindividual nucleic acid molecule within each partition. When enoughpartitions are measured using this technique, the digital informationcan be consolidated to make a statistically relevant measure of startingconcentration for the nucleic acid target (analyte) in the sample.

The concept of digital PCR may be extended to other types of analytes,besides nucleic acids. In particular, a signal amplification reactionmay be utilized to permit detection of a single copy of a molecule ofthe analyte in individual droplets, to permit data analysis of dropletsignals for other analytes in the manner described in Section VII (e.g.,using an algorithm based on Poisson statistics). Exemplary signalamplification reactions that permit detection of single copies of othertypes of analytes in droplets include enzyme reactions.

Qualitative PCR—a PCR-based analysis that determines whether or not atarget is present in a sample, generally without any substantialquantification of target presence. In exemplary embodiments, digital PCRthat is qualitative may be performed by determining whether a packet ofdroplets contains at least a predefined percentage of positive droplets(a positive sample) or not (a negative sample).

Quantitative PCR—a PCR-based analysis that determines a concentrationand/or copy number of a target in a sample.

RT-PCR (reverse transcription-PCR)—PCR utilizing a complementary DNAtemplate produced by reverse transcription of RNA. RT-PCR permitsanalysis of an RNA sample by (1) forming complementary DNA copies ofRNA, such as with a reverse transcriptase enzyme, and (2) PCRamplification using the complementary DNA as a template. In someembodiments, the same enzyme, such as Tth polymerase, may be used forreverse transcription and PCR.

Real-time PCR—a PCR-based analysis in which amplicon formation ismeasured during the reaction, such as after completion of one or morethermal cycles prior to the final thermal cycle of the reaction.Real-time PCR generally provides quantification of a target based on thekinetics of target amplification.

Endpoint PCR—a PCR-based analysis in which amplicon formation ismeasured after the completion of thermal cycling.

Amplicon—a product of an amplification reaction. An amplicon may besingle-stranded or double-stranded, or a combination thereof. Anamplicon corresponds to any suitable segment or the entire length of anucleic acid target.

Primer—a nucleic acid capable of, and/or used for, priming replicationof a nucleic acid template. Thus, a primer is a shorter nucleic acidthat is complementary to a longer template. During replication, theprimer is extended, based on the template sequence, to produce a longernucleic acid that is a complementary copy of the template. A primer maybe DNA, RNA, an analog thereof (i.e., an artificial nucleic acid), orany combination thereof. A primer may have any suitable length, such asat least about 10, 15, 20, or 30 nucleotides. Exemplary primers aresynthesized chemically. Primers may be supplied as at least one pair ofprimers for amplification of at least one nucleic acid target. A pair ofprimers may be a sense primer and an antisense primer that collectivelydefine the opposing ends (and thus the length) of a resulting amplicon.

Probe—a nucleic acid connected to at least one label, such as at leastone dye. A probe may be a sequence-specific binding partner for anucleic acid target and/or amplicon. The probe may be designed to enabledetection of target amplification based on fluorescence resonance energytransfer (FRET). An exemplary probe for the nucleic acid assaysdisclosed herein includes one or more nucleic acids connected to a pairof dyes that collectively exhibit fluorescence resonance energy transfer(FRET) when proximate one another. The pair of dyes may provide firstand second emitters, or an emitter and a quencher, among others.Fluorescence emission from the pair of dyes changes when the dyes areseparated from one another, such as by cleavage of the probe duringprimer extension (e.g., a 5′ nuclease assay, such as with a TAQMANprobe), or when the probe hybridizes to an amplicon (e.g., a molecularbeacon probe). The nucleic acid portion of the probe may have anysuitable structure or origin, for example, the portion may be a lockednucleic acid, a member of a universal probe library, or the like. Inother cases, a probe and one of the primers of a primer pair may becombined in the same molecule (e.g., AMPLIFLUOR primers or SCORPIONprimers). As an example, the primer-probe molecule may include a primersequence at its 3′ end and a molecular beacon-style probe at its 5′ end.With this arrangement, related primer-probe molecules labeled withdifferent dyes can be used in a multiplexed assay with the same reverseprimer to quantify target sequences differing by a single nucleotide(single nucleotide polymorphisms (SNPs)). Another exemplary probe fordroplet-based nucleic acid assays is a Plexor primer.

Label—an identifying and/or distinguishing marker or identifierconnected to or incorporated into any entity, such as a compound,biological particle (e.g., a cell, bacteria, spore, virus, ororganelle), or droplet. A label may, for example, be a dye that rendersan entity optically detectable and/or optically distinguishable.Exemplary dyes used for labeling are fluorescent dyes (fluorophores) andfluorescence quenchers.

Reporter—a compound or set of compounds that reports a condition, suchas the extent of a reaction. Exemplary reporters comprise at least onedye, such as a fluorescent dye or an energy transfer pair, and/or atleast one oligonucleotide. Exemplary reporters for nucleic acidamplification assays may include a probe and/or an intercalating dye(e.g., SYBR Green, ethidium bromide, etc.).

Code—a mechanism for differentiating distinct members of a set.Exemplary codes to differentiate different types of droplets may includedifferent droplet sizes, dyes, combinations of dyes, amounts of one ormore dyes, enclosed code particles, or any combination thereof, amongothers. A code may, for example, be used to distinguish differentpackets of droplets, or different types of droplets within a packet,among others.

Binding partner—a member of a pair of members that bind to one another.Each member may be a compound or biological particle (e.g., a cell,bacteria, spore, virus, organelle, or the like), among others. Bindingpartners may bind specifically to one another. Specific binding may becharacterized by a dissociation constant of less than about 10⁻⁴, 10⁻⁶,10⁻⁸, or 10⁻¹⁰ M. Exemplary specific binding partners include biotin andavidin/streptavidin, a sense nucleic acid and a complementary antisensenucleic acid (e.g., a probe and an amplicon), a primer and its target,an antibody and a corresponding antigen, a receptor and its ligand, andthe like.

Channel—an elongate passage for fluid travel. A channel generallyincludes at least one inlet, where fluid enters the channel, and atleast one outlet, where fluid exits the channel. The functions of theinlet and the outlet may be interchangeable, that is, fluid may flowthrough a channel in only one direction or in opposing directions,generally at different times. A channel may include walls that defineand enclose the passage between the inlet and the outlet. A channel may,for example, be formed by a tube (e.g., a capillary tube), in or on aplanar structure (e.g., a chip), or a combination thereof, among others.A channel may or may not branch. A channel may be linear or nonlinear.Exemplary nonlinear channels include a channel extending along a planarflow path (e.g., a serpentine channel) a nonplanar flow path (e.g., ahelical channel to provide a helical flow path). Any of the channelsdisclosed herein may be a microfluidic channel, which is a channelhaving a characteristic transverse dimension (e.g., the channel'saverage diameter) of less than about one millimeter. Channels also mayinclude one or more venting mechanisms to allow fluid to enter/exitwithout the need for an open outlet. Examples of venting mechanismsinclude but are not limited to hydrophobic vent openings or the use ofporous materials to either make up a portion of the channel or to blockan outlet if present.

Fluidics Network—an assembly for manipulating fluid, generally bytransferring fluid between compartments of the assembly and/or bydriving flow of fluid along and/or through one or more flow pathsdefined by the assembly. A fluidics network may include any suitablestructure, such as one or more channels, chambers, reservoirs, valves,pumps, thermal control devices (e.g., heaters/coolers), sensors (e.g.,for measuring temperature, pressure, flow, etc.), or any combinationthereof, among others.

II. SYSTEM OVERVIEW/ARCHITECTURE

This Section describes the architecture of illustrative systems,including methods and apparatus, for droplet-based assays. The featuresand aspects of the systems disclosed in this Section may be combinedwith one another and/or with any suitable aspects and features ofmethods and apparatus shown and/or described elsewhere in the presentdisclosure. Additional pertinent disclosure may be found in the U.S.provisional patent applications listed above under Cross-References andincorporated herein by reference, particularly Ser. No. 61/277,270,filed Sep. 22, 2009.

A. Exemplary Instrument-Cartridge System for Sample Preparation andAnalysis

FIGS. 2 and 3A show perspective and schematic views, respectively, of anexemplary system 600 for performing droplet-based assays. System 610 maycomprise an instrument 612 and one or more sample cartridges 614 thatconnect to the instrument, to provide sample preparation that isactuated and controlled by the instrument. Sample preparation mayinclude any combination of the processes disclosed in Section III orelsewhere in the present disclosure, such as extraction, purification,lysis, concentration, dilution, reagent mixing, and/or dropletgeneration, among others. Instrument 612 may perform amplification ofnucleic acid in the droplets, detection of signals from the droplets,and data analysis, among others.

Instrument 612 may be equipped with a sample loading region 616, areagent fluidics assembly 618, a thermal cycler 620, a detector 622,control electronics 624 (i.e., a controller), and a user interface 626,among others. The instrument also may include a housing 628, which maysupport, position, fix, enclose, protect, insulate, and/orpermit/restrict access to each other instrument component.

Sample loading region 616 may permit placement of sample cartridges 614into the instrument, generally after a sample has been introduced into aport of each cartridge. The sample loading region may have an openconfiguration for receiving sample cartridges and a closed configurationthat restricts cartridge introduction and removal (e.g., duringinstrument actuation of loaded sample cartridges). For example, thesample loading region may include a tray 630 that is an extendible andretractable and that receives the sample cartridges and positions thecartridges for operational engagement with instrument 612. The tray maybe pulled out manually for loading sample cartridges into the tray andpushed in manually for cartridge operation, or may be coupled to a drivemechanism that drives opening and closing of the sample loading region.

Sample cartridges 614 are depicted in various positions in FIG. 2. Someof the cartridges have been loaded into tray 630, which is extended,while other cartridges are disposed outside instrument 612 (e.g.,stacked, indicated at 632), before or after their use with theinstrument. The sample cartridges may be primed/loaded with one or morefluid reagents before the cartridges are connected to the instrument(e.g., during cartridge manufacture), and/or the sample cartridges maybe primed with one or more fluid reagents supplied by the instrument.Further aspects of sample cartridges that may be suitable for use withinstrument 612 are described elsewhere in the present disclosure,particularly in Section III.

FIG. 3B shows a schematic view of selected aspects of system 610. Thearrows extending across junctions between system components generallyshow directions of fluid or data flow within the system. The linesegments extending across the junctions indicate an electricalconnection and/or signal communication.

Sample cartridges 614 may receive fluid for sample preparation fromreagent fluidics assembly 618. Fluidics assembly 618 may include reagentcartridges or containers 634 (also see FIG. 2), which may be disposableand/or reusable (i.e., refillable). Fluidics assembly 618 also mayinclude sample cartridge fluidics 636, which, in conjunction with afluidics controller and injector 638, enable controlled fluid flow. Forexample, fluid may flow from the reagent cartridges to the samplecartridges, may flow within each sample cartridge, and/or may flow fromeach sample cartridge to thermal cycler 620 as droplets disposed in animmiscible carrier fluid.

Thermal cycler 620 may subject the droplets to thermal cycles thatpromote amplification, in preparation for detection of droplet signalsby detector 622. Further aspects of thermal cyclers and detectors aredescribed elsewhere herein, such as in Sections V and VI. Afterdetection, the droplets and carrier fluid may flow to a waste receptacle640.

Data from detector 622 may be communicated to control electronics 624.The control electronics may analyze the data (e.g., as described inSection VII), and communicate the data to user interface 626, amongothers. The control electronics also may receive input data, such aspreferences, instructions, and/or commands, from the user interface. Thecontrol electronics may be in communication with and/or may beprogrammed to control any other aspects of system 600. For example, thecontrol electronics may be in communication with cartridges 614. In someembodiments, each cartridge may be a “smart cartridge” that carries amemory device 627. The memory device may be readable by the controller,and, optionally, writable, too. The memory device may carry informationabout the cartridge, such as reagents pre-loaded to the cartridge, dataabout the loaded sample, aspects of sample processing performed by thecartridge, or any combination thereof, among others. The controlelectronics also may be connected to an external communication port 642,which also may provide data input/output. A power supply 644 (e.g., aline or battery power source) may provide power to the controlelectronics. The power may be conditioned by any suitable element(s)(e.g., a rectifier) between the power supply and the controlelectronics.

B. Exemplary Instrument for Analysis of Pre-Prepared Samples

FIG. 4 shows another exemplary system constructed as an instrument 650for performing droplet-based assays. Instrument 650 may be capable ofperforming droplet-based assays of nucleic acid amplification, generallyas described above for system 610. However, instrument 650 may bedesigned to process and analyze samples that are supplied as pre-formedemulsions or prepared samples (e.g., purified nucleic acids that are notyet in emulsion form).

Instrument 650 may be equipped with a sample loading region 652, areagent fluidics assembly 654, a thermal cycler 656, a detector 658,control electronics 660 (i.e., a controller), a user interface 662, anda housing 664, among others, which each may function generally asdescribed above for system 610. However, sample loading region 652 andreagent fluidics assembly 654 may differ from the analogous structuresin instrument 612. In particular, the sample preparation proceduresperformed in the sample cartridges of system 610 (see FIG. 2) areperformed outside of instrument 650, before sample loading.

Sample loading region 652 may include a tray 666 and an array ofcompartments or reservoirs 668, such as wells. Reservoirs 668 may beprovided by a plate 670, such as a microplate, which may be receivedand/or supported by the tray. Plate 670 may be removable, to permitplacing samples into reservoirs 668 while the plate is spaced from theinstrument. Alternatively, or in addition, samples may be placed intoreservoirs 668 while the reservoirs are supported by thetray/instrument. In some examples, plate 670 may be a droplet generatorplate (e.g., see below in this Section and Sections III and IV). Ifstructured as a droplet generator plate, the plate may generate dropletsbefore or after the plate is loaded into instrument 650.

Each reservoir may receive a pre-prepared sample. The pre-preparedsample may or may not be in emulsion form. If not in emulsion form, thesample may have been processed before loading into the reservoir (e.g.,processed by extraction, purification, lysis, concentration, dilution,reagent mixing, or any combination thereof), to ready the sample fordroplet generation. Alternatively, the sample may be a pre-formedemulsion of droplets in an immiscible carrier fluid. The emulsion may beformed prior to loading the sample into the reservoir by partitioninginto droplets an assay mixture that includes a sample and at least onereagent. Each droplet thus may contain a partition of the sample.Droplet packets from the emulsions may be transported serially or inparallel from reservoirs 668 to at least one thermal cycler 656 of theinstrument.

User interface 662 of instrument 650 may (or may not) be different inconfiguration from user interface 626 of system 610 (compare FIGS. 2 and4). For example, user interface 662 may be spaced from the body ofinstrument 650 (e.g., disposed outside of and spaced from housing 664).User interface 662 may be in wired or wireless communication withcontrol electronics 660 of the instrument.

C. Overview of Droplet-Based Assay Systems

FIG. 5 shows a flowchart 680 listing exemplary steps that may beperformed in a method of sample analysis using droplet-based assays. Thesteps listed may be performed in any suitable combination and in anysuitable order and may be combined with any other step(s) of the presentdisclosure.

At least one sample may be loaded, indicated at 682. The sample may beloaded by placing the sample into a port (e.g., a well, chamber,channel, etc.) defined by any of the system components disclosed herein.The sample may be loaded in any suitable form, such as unlysed or lysed,purified or crude, pre-mixed with reagent or not pre-mixed, diluted orconcentrated, partitioned into droplets or non-partitioned, or the like.In some cases, a plurality of samples may be loaded into respectiveports and/or into an array of reservoirs.

The sample may be processed, indicated at 684. Any suitable combinationof sample processing steps may be performed after (and/or before) sampleloading to prepare the sample for droplet generation. Exemplaryprocessing steps are described in Section III.

Droplets may be generated from the sample, indicated at 686. Forexample, droplet generation may be performed after the sample has beenmodified by mixing it with one or more reagents to form a bulk assaymixture. Droplet generation may divide the bulk assay mixture into aplurality of partitioned assay mixtures (and thus sample partitions)that are isolated from one another in respective droplets by anintervening, immiscible carrier fluid. The droplets may be generatedfrom a sample serially, such as from one orifice and/or one dropletgenerator (which may be termed an emulsion generator). Alternatively,the droplets may be generated in parallel from a sample, such as fromtwo or more orifices and/or two or more droplet generators in fluidcommunication with (and/or supplied by) the same sample. As anotherexample, droplets may be generated in parallel from a perforated platedefining an array of orifices. In some examples, the droplets may begenerated in bulk, such as by agitation or sonication, among others. Insome examples, a plurality of emulsions may be generated, eitherserially or in parallel, from a plurality of samples.

Droplets may be loaded (i.e., introduced) into a reaction site (alsotermed a reactor), indicated at 688. The droplets may be loaded by flowtransport, which may be continuous or stopped one or more times. Thus,the droplets may (or may not) be stored, indicated at 690, at one ormore discrete storage sites, after their generation and before loadinginto the reaction site. Alternatively, the droplets may be loaded into areaction site without substantial flow, for example, with the dropletscontained by a vessel that is moved to the reaction site. In otherexamples, the droplets may be generated at the reaction site (e.g.,inside a thermal cycler). In any event, after droplet generation,droplets may be placed into a reaction site with the droplets disposedin a vial (or other vessel), a reaction channel (e.g., in tubing), animaging chamber/flow cell with a high aspect ratio, or the like. Furtheraspects of droplet manipulation, such as selection fortransport/loading, transport, storage, routing, pre-processing (e.g.,heating), and concentration are described below in this Section.

A “reaction site” is a region where droplets are subjected to conditionsto promote one or more reactions of interest, such as nucleic acidamplification. Accordingly, a reaction site may provide one or moretemperature-controlled zones of fixed or varying temperature (and/orother physical conditions) suitable for a particular reaction(s) to beperformed and/or promoted in the droplets. The reaction site may be aflow-through site, where the droplets are subjected to fixed or varyingreaction conditions while flowing through at least one channel or may bea static site where the droplets are subjected to fixed or varyingreaction conditions while the droplets are disposed in a stationaryvolume of fluid (i.e., not flowing). An exemplary reaction site, namely,a flow-based thermal cycler, is included in many of the exemplarysystems of this Section and is described in more detail in Section V.

Droplets may be “reacted,” indicated at 692. More specifically, thedroplets may be subjected to one or more suitable reaction conditions ina reaction site, according to the type of assay mixture(s) contained bythe droplets, such that components of the droplets, or the dropletsthemselves, undergo a desired reaction (or change of state). Forexample, the droplets may be subjected to thermal cycling (or may beprocessed isothermally) for amplification assays, such as any of theassays described in Section I, among others.

Reaction of droplets generally subjects the droplets to one or moreconditions that promote at least one binding and/or chemical reaction ofinterest in the droplets. Reaction of droplets also generally subjectsthe droplets to each condition for a predefined period (or periods) oftime, which may be fixed or variable, and may be repeated. The dropletsmay be subjected to two or more conditions serially or in parallel, andonce or a plurality of times, for example, cyclically. Exemplaryconditions include a temperature condition (i.e., to maintain droplettemperature, heat droplets, and/or or cool droplets), exposure to light,variations in pressure, or the like.

Droplets may be reacted by flow through a reaction site, in a “flowreaction.” Droplets may be subjected to at least one condition that isuniform or that varies spatially along a flow path through the reactionsite. For example, the temperature along the flow path may varyspatially, to heat and cool droplets as the droplets follow the flowpath. In other words, the reaction site may include one, two, or moretemperature-controlled zones of at least substantially fixed temperaturethat the droplets travel through. Further aspects of flow-throughreaction sites with fixed temperature zones and thermal cycling aredescribed elsewhere herein, such as in Section V, among others.

Droplets alternatively may be reacted while disposed in a static volumeof fluid, that is, without substantial fluid flow, in a “staticreaction.” For example, the droplets may react while disposed in a wellor a chamber, among others. In this case, the droplets may be subjectedto a fixed condition during the reaction (e.g., a fixed temperature foran isothermal reaction), or to a variable condition that variestemporally (i.e., with respect to time) during the reaction (without therequirement for the droplets to move). For example, the droplets may beheld in a temperature-controlled zone that changes in temperature overtime, such as cyclically to perform PCR. In any event, static reactionsmay permit batch reaction of arrays of emulsions in parallel, such as inbatch amplification of emulsions.

Droplets may be detected, indicated at 694. Detection may be performedserially while the droplets are flowing (i.e., flow-based or dynamicdetection). Alternatively, detection may be performed with the dropletsdisposed in a static volume of fluid (i.e., static detection, such aswith flow stopped (i.e., stopped-flow detection)). In some examples,static detection (or dynamic detection) may include imaging a set ofsubstantially static (or flowing) droplets, which may be arrangedgenerally linearly or in a plane, to obtain an image of the droplets.Further aspects of detection, including flow-based and stopped-flowdetection are described elsewhere herein, such as in Section VI, amongothers.

Dynamic/static modes of reaction and detection may be combined in anysuitable manner. For example, flow-based reaction of droplets may becombined with flow-based detection or stopped-flow detection (e.g.,imaging) of the droplets. Alternatively, static reaction of droplets,such as batch amplification of emulsions, may be combined withflow-based detection or static detection (e.g., imaging) of thedroplets.

Data detected from the droplets may be analyzed, indicated at 696. Dataanalysis may, for example, assign droplet signals as positive ornegative for amplification of a nucleic acid target (or two or moretargets in a multiplexed reaction), may determine a number and/orfraction of the droplets that are positive for amplification, mayestimate a total presence (e.g., concentration and/or number ofmolecules) of the nucleic acid target in the sample, or the like.Further aspects of data analysis are described elsewhere herein, such asin Sections VII and VIII, among others.

FIG. 6 shows selected portions of an exemplary system 700 for performingdroplet-based assays. Any one component or combination of the depictedsystem components may be omitted from the system, and any additionalcomponents disclosed elsewhere herein may be added to the system. Thearrows indicate an exemplary sequence in which sample, droplets, and/ordata may move between structural components of the system. However, eachof the structural components may be used more than once with the samedroplets, and/or may be utilized in a different sequence than shownhere.

System 700 may include one or more of any or each of the followingcomponents: a sample processor 702 (also termed a sample processingstation), a droplet generator 704, a droplet transporter 706, a reactionsite (or reactor) 708 (also termed a reaction station (e.g., a heatingstation, which may heat or heat and cool)), a detector 710 (also termeda detection station), and a controller 712, among others. Anycombination of the components may be connected to one anotherphysically, fluidically, electrically, and/for signal transfer, amongothers.

The components may operate as follows, with reference to steps of method680 (FIG. 5). Sample processor 702 may receive a sample to be analyzed,such as a sample that is loaded in step 682, and may process the samplein the manner described above for step 684. Droplet generator 704 maygenerate droplets as described for step 686. Droplet transporter 706 mayload the droplets generated, as described for step 688, and thus mayprovide selectable transport/loading, transport, storage (step 690),routing, pre-processing (e.g., heating), and concentration, amongothers, of the generated droplets. Reaction site 708 may enable a flowreaction or a static reaction of the loaded droplets, and detector 710may provide dynamic or static detection of droplets, as described forstep 694. Controller 712 may analyze data received from detector 710, asdescribed for step 696. Also, controller 712 may be in communicationwith and/or may be programmed to control any suitable combination ofsystem components, as indicated by dashed lines extending from thecontroller to each other system component. Controller also may contain acomputer-readable medium (e.g., a storage device, such as a hard drive,CD-ROM, DVD-ROM, floppy disk, flash memory device, etc.) includinginstructions for performing any of the methods disclosed herein.

D. Exemplary System with Flow-Based Amplification

FIG. 7 shows a schematic view of an exemplary system 720 with flow-basedamplification and with droplet loading that is decoupled from dropletgeneration. Any one component or combination of the depicted systemcomponents may be omitted from the system, and any additional componentsdisclosed elsewhere herein may be added to the system. The solid arrowsindicate an exemplary sequence in which sample 722, reagent 724, anddroplets 726 may move between structural components of the system. Thevertical dashed arrows above and below various system componentsindicate optional addition (e.g., inflow) and/or removal (e.g., outflow)of an immiscible carrier fluid (e.g., oil) and/or waste with respect tothese components.

System 720 may include a mixer 728 and a droplet generator 730. Mixer728 may receive a sample 722 and at least one reagent 724 and combinethem to form an assay mixture. The mixer may be an automated device, ormixing may be performed manually by a user, such as by bulk mixing,before loading the assay mixture into the droplet generator. Dropletgenerator 730 may receive the assay mixture from the mixer and generatean emulsion 732 of droplets 726 in an immiscible carrier fluid 734, suchas oil that is introduced into the droplet generator, indicated at 736,at the same time as the assay mixture. Formation of droplets 726 may bedriven by pressure and/or pumping, indicated at 738. In some examples,the droplet generator may function as the mixer by generating dropletsfrom confluent streams of sample and reagent. Waste fluid also may exitthe droplet generator, indicated at 740.

System 720 may have any suitable number of droplet generators. Thedroplet generators may be used to generate any suitable number ofseparate, distinct emulsions from one sample or a plurality of samples,and from one reagent or a plurality of reagents (e.g., reagents fordifferent species of nucleic acid target). Exemplary mixers and dropletgenerators are described in Sections III and IV.

Emulsion 732 or a set of distinct emulsions may be stored in at leastone storage site 742 or in a plurality of such sites before droplets ofthe emulsion(s) are reacted. As a result, droplet generation may bedecoupled from reaction of the droplets. The storage site may, forexample, be a well, a chamber, a tube, or an array thereof, such asformed by a plate (e.g., a microplate).

System also may include a serial arrangement of a droplet transportportion 744, (also termed a droplet transporter) and a thermal cycler746. Transport portion 744 may include a droplet pick-up or intakeregion 748 that forms an inlet at which droplets 726 are transferredfrom storage site 742 into the transport portion. Transport portion 744also may include a droplet loader 750 that sends droplets to thermalcycler 746. The transport portion also may include one or more storagesites 752 for storing droplets after they have been transferred intotransport portion 744.

In some examples, the transport portion also may be capable of loadingdroplets more directly to the detector, without sending them first tothe thermal cycler. In particular, system 720 may include a bypasschannel 753 or bypass pathway that connects transport portion 744 to thedetector without travel through the thermal cycler. The system mayinclude one or more valves that can be operated to send droplets eitherto bypass channel 753 or to thermal cycler 746. The use of bypasschannel 753 may, for example, permit more rapid calibration of systemcomponents, because calibration droplets can travel to the detectorfaster if thermal cycling is omitted. Section VIII describes furtheraspects of the use of a bypass channel and calibration droplets.

Carrier fluid and/or waste fluid optionally may be removed from storagesite 742, droplet pick-up region 748, and/or droplet loader 750,indicated respectively at 754-758. Alternatively, or in addition,carrier fluid may be added to the droplet pick-up region, indicated at759, and/or the droplet loader, indicated at 760, such as to facilitatedriving droplets into thermal cycler 746 and/or to flush droplets fromthe pick-up region and/or droplet loader.

An emulsion including droplets 726 may flow through (a) thermal cycler746, (b) at least one detection site (e.g., a detection channel/chamber)adjacent at least one detection window 762 that is operatively disposedwith respect to detector 764, and (c) through an oil recovery region 766and then to a waste receptacle. One or more valves 770 may be disposedgenerally between the thermal cycler and the detector, to providecontrol of emulsion flow downstream of the thermal cycler, with respectto the at least one detection channel/chamber. For example, valves 770may be operated to stop flow of droplets adjacent to the detectionwindow and/or to switch flow of the emulsion between two or moredetection windows (e.g., see Section VI). Carrier fluid may be removedfrom the emulsion and/or introduced into the emulsion in or near thermalcycler 746 and/or detector 764, indicated respectively at 772, 774.Removal of carrier fluid may, for example, provide a more concentratedemulsion for detection. Introduction of carrier fluid may, for example,provide flow-focusing of droplets within a detection channel and/or withrespect to the detection window (e.g., see Section VI). Alternatively,or in addition, droplets may be sent to a waste receptacle, indicated at775, for collection from the thermal cycler, without traveling through adetection station.

Carrier fluid also may be removed from the flow stream by oil recoveryregion 766, indicated at 776. Removal may be effected by any suitablemechanism, such as pillars, at least one membrane, one or moreoil-selective side channels, gravity separation, or the like.

E. Overview of Droplet Manipulation

FIGS. 8-10 provide an overview of droplet manipulation, includingmethods and apparatus, emphasizing droplet transport and exemplary typesof droplet manipulation that may be performed in connection therewith(e.g., storage, concentration, selection, etc.).

FIG. 8 shows a flowchart 810 listing exemplary steps that may beperformed in an exemplary method of sample analysis using droplet-basedassays in which droplets are transported from a droplet generator and/ora droplet reservoir to a reaction site. The steps listed may beperformed in any suitable combination and in any suitable order and maybe combined with any other suitable step(s) of the present disclosure.

Droplets may be generated, indicated at 812. The droplets may begenerated serially, in parallel, or in bulk. Further aspects of dropletgeneration are disclosed elsewhere herein, such as in Sections III andVI, among others.

The droplets, optionally, may be stored, indicated at 814. A set ofdroplets (e.g., an emulsion) may be stored in a droplet reservoir. Insome examples, two or more distinct sets of droplets may be stored intwo or more respective reservoirs, such as in an array of emulsions. Insome examples, storage of the droplets may be omitted.

The droplets, optionally, may be concentrated, indicated at 816.Concentrating droplets (also termed concentrating an emulsion) resultsin an increase in the number of droplets per unit volume of emulsion andincreases the volume fraction occupied by the droplets in an emulsion.Concentration of an emulsion may be conducted before, during, and/orafter droplet storage.

One or more of the droplets (including one or more packets of droplets)may be transported to a reaction site, indicated at 818. Transport maybe achieved by continuous flow, or by flow initiated selectably in oneor more discrete stages, after droplet generation and/or initial dropletstorage. The droplets may be reacted at the reaction site, indicated at820.

Signals may be detected from droplets of the packet, indicated at 822.For example, one or more measurements may be performed on one or aplurality of the droplets during and/or after reaction of the droplets.Further aspects of droplet detection are disclosed elsewhere herein,such as in Section VI, among others.

FIG. 9 shows a flowchart 830 listing exemplary steps that may beincluded in a step of transporting droplets (i.e., step 818) in themethod of FIG. 8.

A droplet reservoir (also termed an emulsion reservoir) may be selected,indicated at 832. The droplet reservoir may be selected from an array ofdroplet reservoirs holding distinct emulsions and/or distinct assaymixtures. Selection may be performed by a controller, by a user, or acombination thereof.

Droplets from the selected reservoir may be transferred to a droplettransporter, indicated at 834. The transferred droplets may be referredto as a packet. In some examples, a plurality of reservoirs may beselected and a plurality of droplet packets from respective selectedreservoirs may be transferred serially (or in parallel) to the droplettransporter.

The packet(s) of droplets, optionally, may be held (i.e., stored) by thedroplet transporter, indicated at 836. Droplets may be stored by thedroplet transporter by stopping flow of the droplets, such as byisolating the droplets from a flow stream traveling to the reactionsite. Accordingly, the droplets may be held in static (non-flowing)fluid (i.e., without substantial net flow of the continuous phase).

The packet of droplets, or at least a portion thereof, may be loadedinto a reaction site (e.g., a thermal cycler), indicated at 838, whichmay be described as the droplets being sent or introduced into thereaction site. Packets of droplets may be loaded serially.Alternatively, packets of droplets may be loaded in parallel, such asloaded into distinct thermal cyclers or into separate flow paths throughthe same thermal cycler. In some examples, the step of holding dropletsmay be omitted, such that transfer of a packet of droplets from thereservoir and loading the packet into a reaction site occur bycontinuous flow.

FIG. 10 shows selected portions of an exemplary system 850 capable ofperforming the method of FIG. 8. The arrows indicate an exemplarysequence in which droplets may move between structural components of thesystem. However, each of the structural components may be optional, maybe used more than once with the same packet of droplets, and/or may beutilized in a different sequence than shown here.

System 850 may incorporate at least one droplet generator 852, at leastone droplet reservoir 854, at least one droplet transporter 856, atleast one reaction site 858 (also termed a reaction region or dropletprocessing assembly), and at least one detector 860. All or any subsetof these structural components may be connected to one another, with anysuitable relative spatial relationships, to form an instrument or aninstrument-cartridge assembly (e.g., see FIGS. 2-4). In some examples,one or more of the system components may be utilized remotely, such as adroplet generator that forms droplets (and/or a droplet reservoir thatstores droplets) while the droplet generator is not connected to thetransporter, reaction site, and/or detector. System 850 also may beequipped with at least one controller 862, which may be in communicationwith and/or may be programmed to control any suitable combination ofsystem components, as indicated by dashed lines extending from thecontroller to each other system component.

Droplets formed by droplet generator 852 may be transported by droplettransporter 856, after droplet formation, to reaction site 858, topromote one or more reactions, and to detector 860, to provide detectionof droplet signals. Before and/or during their transport, the dropletsmay be received by at least one droplet reservoir 854 or serially (or inparallel) by two or more droplet reservoirs, and then stored in thedroplet reservoir(s) for an adjustable (and selectable) period of time.Droplet storage is an optional part of the system and thus the dropletreservoir may be omitted.

Any suitable droplet generator(s) 852 and detector(s) 860 may beincorporated into the system, such as any of the droplet generatorsand/or detectors disclosed herein (e.g., see Sections III, IV, and VI).

A “droplet reservoir,” also termed a “storage site” or “emulsionreservoir,” is any compartment where droplets can be stored, generallyin a static volume of fluid, and then accessed at a selectable time. Thedroplet reservoir may be a well, a chamber, or the like. Exemplarydroplet reservoirs may be provided as an array of isolated or isolatablestorage sites, such as an array of wells or chambers, among others. Thearray of storage sites may be provided by a plate.

Droplet transporter 856 may be composed of one or more structures and/orone or more devices that provide selectable transport of droplets fromat least one droplet generator and/or at least one droplet reservoir toa reaction site. Selectable transport may permit selection of thedifferent droplet packets sent to a reaction site, the order in whichthe droplet packets are sent, the time at which each droplet packet issent, etc. Different droplet packets may have different sample-reagentcombinations, different droplets sizes, different sample and/or reagentdilutions, etc. In any event, the selection may be performed by acontroller, a user, or a combination thereof. For example, the selectionmay be based on an order selected by a user and/or programmed into thecontroller, an arbitrary order selected by the controller, or a dynamicorder determined in real time by the controller based on one or moreassay results obtained by the system, or a combination thereof, amongothers.

F. Exemplary Droplet Transporter

FIG. 11 shows selected aspects of an example 868 of droplet transporter856 (FIG. 10). Transporter 868 may incorporate any combination of atleast one intake conduit 870, at least one outflow conduit 872, at leastone storage site 874, 876, one or more pumps 878 and/or pressuresources/sinks, and/or one or more valves 880 (e.g., 2-way, 3-way, 4-way,and/or multi-position valves and/or injection loops), among others. Thetransporter also may include one or more unions, tees, crosses,debubblers, or any combination thereof, among others.

Intake conduit 870 may be configured to receive droplets 881 by pickingup and/or taking in droplets from a droplet reservoir 882 (orcontinuously from a droplet generator). Thus, the intake conduit mayabut and/or extend into the droplet reservoir, to provide contact withan emulsion 884 containing the droplets, such that fluid can flow fromthe emulsion into the intake conduit. The intake conduit may bedescribed as a needle, a tip, a tube, or a combination thereof, amongothers, and may be sized in cross-section to receive droplets in singlefile or multiple file (side-by-side).

Outflow conduit 872 may be joined directly to the intake conduit or maybe separated from the intake conduit by one or more valves 880, storagesites 874, 876, or the like. For example, in FIG. 11, the intake andoutflow conduits are separated by three valves 880 and two storage sites(874, 876).

Each pump 878 (and/or positive/negative pressure source/sink) may drivefluid flow through the intake conduit and/or the outflow conduit, and/orto and/or from the holding site(s). The pump also may drive fluidthrough a reaction site 885, or a distinct pump may be used for thispurpose. In some examples, droplet transporter 868 may include at leastone pump (or pressure source/sink) to transfer droplets into thetransporter and at least one other pump (or pressure sources/sink) todrive droplets out of the transporter for droplet loading into reactionsite 885.

Each storage site 874, 876 may be connected to intake conduit 870 andoutflow conduit 872, to permit fluid flow between these structures. Forexample, valves 880 may provide selectable and adjustable fluidcommunication between intake conduit 870, outflow conduit 872, and thestorage sites. The valves also may permit fluid to be sent, indicated at886, from either storage site 874, 876 to a waste port.

Droplet transporter 868 may include any other suitable elements. Forexample, the transporter further may be equipped with a drive assembly887 that drives relative movement of intake conduit 870 with respect todroplet reservoir 882, in one, two, or three dimensions. For example, anarray 888 of droplet reservoirs (e.g., a plate with wells) may beconnected to and/or supported by a stage or other support member 890that is driven in x-, y-, and z-directions, to permit selectableplacement of the intake conduit into each of the reservoirs of thearray/plate, in any order. In other examples, the droplet reservoirs mayremain stationary while the intake conduit is driven into contact withthe contents of selected reservoirs. Droplet transporter 868 also oralternatively may incorporate at least one heater 892, which may bepositioned to apply heat to any suitable portion (or all) of the droplettransporter, such as droplet reservoirs 882, intake conduit 870, one ormore storage sites 874, 876, outflow conduit 872, or any combinationthereof, among others. Application of heat may pre-process the droplets,prior to loading the droplets into the reaction site, such as to promotean enzyme reaction (e.g., reverse transcription), to activate a reagent(e.g., an enzyme such as in a hot start prior to an amplificationreaction; see Section V), or the like.

The droplet transporter (and/or any other portion of system 850) furthermay include at least one packing feature 894 to increase theconcentration of droplets. The packing feature may increase the volumefraction of an emulsion occupied by droplets, which may, for example, bedesirable to decrease the amount of energy spent on heating carrierfluid, to increase the rate at which droplets may be detected by aflow-based (serial) detector, and/or to increase the number of dropletsthat may be detected simultaneously by an imaging detector, amongothers. A suitable concentration of droplets (i.e., the “packingdensity”) may be achieved during droplet generation or the packingdensity may be increased after droplet generation. An increase inpacking density may be achieved by removing carrier fluid from anemulsion, while the emulsion is static (e.g., during storage) orflowing, and/or by selective intake of droplets from a stored emulsion,among others. Droplets may be concentrated locally in a stored emulsionby (1) centrifugation, (2) gravity coupled with a density differencebetween the droplets and the carrier fluid (i.e., the droplets float orsink in the carrier fluid), (3) electrokinetic concentration ofdroplets, (4) magnetic concentration of droplets, or the like. Thepacking density may be increased during flow by using one or more sidevent lines of smaller diameter (or one or more membranes) thatselectively permit lateral flow (and removal) of carrier fluid.Alternatively, or in addition, the packing density may be increasedduring fluid flow by utilizing droplet inertia.

G. Exemplary System with Coupled Droplet Generation and Transport

FIG. 12 shows a continuous flow example 910 of system 850 (see FIG. 10)in which droplet generation and droplet transport to a reaction site arecoupled by continuous flow such that droplets are not stored. System 910may comprise a serial arrangement of a droplet generator 912, a droplettransport region 914, a thermal cycler 916, a detector 918, and awaste/collection reservoir 920. Droplet generator 912 may be supplied bya carrier fluid, such as oil 922, and a non-partitioned assay mixture924 of sample and reagent. The oil and the assay mixture each may bedriven to droplet generator 912 by a respective pump or pressure source926, 928. Here, the droplet generator is structured as a cross, but anyother configuration may be suitable (e.g., see Sections III and IV).Droplets 930 formed by the droplet generator may flow continuouslythrough droplet transport region 914 to thermal cycler 916, due tocontinuous fluid flow driven by pumps 926, 928. In other examples, oneor more additional pumps or pressure sources/sinks may be used to driveflow through the thermal cycler.

H. Exemplary Systems with Decoupling of Droplet Generation and Transport

FIGS. 13 and 14 show exemplary systems with decoupling of dropletgeneration and transport.

FIG. 13 shows an example 940 of system 850 in which droplet generationand droplet transport to a reaction site are decoupled. System 940 mayinclude a droplet reservoir 942 holding an emulsion 944 of preformeddroplets 946 in a carrier fluid 948. Droplets 946 may be formed off-linefrom downstream portions of system 940. The droplets, when formed by atleast one droplet generator, may flow continuously into dropletreservoir 942. Alternatively, the droplets may be transferred into thedroplet reservoir with a fluid transfer device (e.g., a pipette orsyringe) from another storage site at a selectable time after dropletgeneration. In any event, droplet reservoir 942 may be placed intoconnection with downstream components of system 940 after (or before)droplet formation, permitting droplets 946 to be stored for anadjustable, selectable period of time after (and, optionally, before)the droplet reservoir becomes connected to the downstream systemcomponents.

System 940 may incorporate a serial arrangement of a droplet transportregion 950, a thermal cycler 952, a detector 954, and at least onepressure source/sink, such as a downstream pressure sink (e.g., syringepump 956), an upstream pressure source 958, or both. Droplet transportregion 950 may include an intake conduit 960 that extends into dropletreservoir 942 and into contact and fluid communication with emulsion944. Droplets 946 may be drawn into the intake conduit as a result of anegative pressure exerted by a downstream vacuum source (or pressuresink) 956 (e.g., a syringe pump), and/or a positive pressure exerted onemulsion 944 by an upstream pressure source 960 (e.g., another pump),among others. As shown here, the droplets may be dispersed non-uniformlyin the emulsion, for example, concentrated selectively toward the top orthe bottom of the emulsion by gravity, centrifugation, magneticattraction, electrokinetic motion, and/or the like, to permit removal ofdroplets at a higher packing density than the average packing density inthe emulsion. Alternatively, or in addition, the carrier fluid may beremoved selectively (e.g., removed and discarded) where the dropletpacking density is lower than average. In any event, droplets 946 may bedriven by continuous flow from the emulsion, through transport region950 and thermal cycler 952, past detector 954, and into a reservoir 962provided by syringe pump 956.

FIG. 14 shows an example 970 of system 850 that is generally related tosystem 940 of FIG. 13, with selected components replicated such thatsystem 970 is capable of transporting, reacting, and/or detecting aplurality of droplet packets in parallel. System 970 may include aserial arrangement of an emulsion array 972, a droplet transporter 974,a thermal cycler 976, one or more detectors 978, and one or more pumpsor pressure sources/sinks, such as a syringe pump 980.

Emulsion array 972 may include emulsions 982 held in an array of dropletreservoirs 984 formed by a plate 986. The emulsions may be formedseparately from the plate and then transferred to the plate.Alternatively, the plate may be a droplet generator plate incorporatingan array of droplet generators 988, which form the emulsions containedin droplet reservoirs 984. Further aspects of droplet generator platesare disclosed below in this Section and in Sections III and IV.

Droplet transporter 974 may include a line of intake conduits or needles990 for intake of droplets in parallel from a row of droplet reservoirs984 of plate 986. The tips of intake conduits 990 may be spaced to matchthe spacing of droplet reservoirs 984 in each row of the plate. Droplettransporter 974 also may include a drive assembly 992 that drivesrelative movement of plate 986 and intake conduits 990 in at least twodimensions or in three dimensions. In particular, operation of the driveassembly may place the intake conduits serially into fluid communicationwith each row of emulsions, in a predefined or selectable order. Inother examples, the droplet transporter may include a three-dimensionalarray of intake conduits, which may be arranged in correspondence withthe rows and columns of droplet reservoirs formed by plate 986, topermit parallel uptake of droplets from two or more rows of dropletreservoirs (e.g., all of the droplet reservoirs in parallel). With anyarrangement of intake conduits, each intake conduit may be connected toa respective valve. Operation of the valve may determine whether anintake conduit is active or inactive for droplet intake. Alternatively,the intake conduits may be connected to the same multi-position valve,which may be operated to select only one of the intake conduits fordroplet intake at a time, to provide serial intake of droplets fromdroplet reservoirs.

Droplet intake may be driven by one or more pumps. For example, anegative pressure applied by syringe pump 980 may draw droplets intointake conduits 990. Alternatively, or in addition, a positive pressureapplied by a positive pressure source, such as a pump 994 of droplettransporter 974, may push droplets into the intake conduits, in a manneranalogous to that described for system 940 of FIG. 13. In particular,pump 994 may be connected to droplet transporter 974 via a manifold 996.Each intake conduit may extend through the manifold in a sealedrelationship with the manifold. The manifold may be movable into asealed relationship with each row of droplet reservoirs, by operation ofdrive assembly 992, to form a sealed chamber 998 over each row serially.Accordingly, pump 994 may pressurize the chamber to urge droplets fromthe reservoirs of a row in parallel into the intake conduits.

Thermal cycler 976 may include a plurality of reaction channels providedby coiled tubes 1000-1014 each forming a separate, respective connectionwith a different intake conduit 990. The coiled tubes may follow agenerally helical path interspersed with one another. For example, thetubes may be braided together and/or wrapped collectively. In any event,droplet transporter 974 may load packets of droplets into the coiledtubes in parallel, and the packets may be thermally cycled in parallel,while following separate flow paths. Droplets from each coiled tube alsomay be detected in parallel, indicated at 1016, by detector 978. Inother examples, each intake conduit 990 may be connected to arespective, distinct thermal cycler, or intake conduits 990 may feeddroplets into the same coiled tube or other reaction channel.

I. Exemplary Decoupled System Utilizing an Autosampler

FIGS. 15 and 16 show an exemplary system combining decoupling of dropletgeneration and transport with autosampling.

FIG. 15 shows another example 1030 of system 850 of FIG. 10 in whichdroplet generation and droplet transport to a reaction site aredecoupled. System 1030 may incorporate a serial arrangement of areservoir array 1032, a droplet transporter 1034 comprising anautosampler 1036, a reaction site 1038 (e.g., a thermal cycler 1040), adetector 1042, and a waste/collection reservoir 1044. Droplets maytravel from array 1032 to reaction site 1038 through the action ofautosampler 1036, may be detected by detector 1042 during/afterreaction, and then may be collected after detection by reservoir 1044.

Reservoir array 1032 may be structured as a plate 1046 providing anarray of droplet reservoirs, such as wells 1048, each containingdroplets 1050. Accordingly, plate 1046 may be structured as a dropletgenerator plate having any combination of the features describedelsewhere herein. Alternatively, plate 1046 may hold droplets that weregenerated separately from the plate and then transferred to the wells ofthe plate.

Autosampler 1036 generally includes any device or assembly of devicesthat provides serial intake of fluid into a conduit (e.g., an intakeconduit) from an array of reservoirs. The autosampler generally iscapable of picking up droplets from any reservoir or sequence ofreservoirs of the array and may be controllable to intake a variablevolume of fluid from each reservoir. The autosampler may include aneedle 1052 that serves as an intake conduit, one or more pumps orpressure sources/sinks 1054, one or more valves 1056, or any combinationthereof, among others. The autosampler may include a drive assembly 1058that controllably drives motion of needle 1052 in three dimensions, suchas along three orthogonal axes. For example, the drive assembly maypermit the needle to be positioned in an x-y plane over any selectedreservoir 1048, and then to be moved along a z-axis, to move the needleinto contact with fluid in the selected reservoir, for droplet intake,and then out of contact with the fluid, for movement to anotherreservoir (or for intake of air). In other examples, the drive assemblymay drive movement of the array of reservoirs while the needle remainsstationary. In other examples, there may be a z-axis drive assembly todrive z-axis motion of the needle, and an x-y axis drive assembly todrive x-y motion of the array of reservoirs, or vice versa.

FIG. 16 shows selected portions of system 1030 of FIG. 15, with needle1052 of autosampler 1036 picking up droplet packets 1060-1064 from acorresponding respective series of wells 1066-1070 of plate 1046.Adjacent droplet packets may be separated from one another inautosampler 1036 by any suitable spacer region 1072. The spacer regionmay contain one or more segments 1074 of one or more spacer fluids. Forexample, a spacer liquid 1076 may be disposed in a well 1078 of thearray or in another accessible reservoir. Needle 1052 may move to well1078, to take in spacer liquid 1076, after each droplet packet is pickedup. Alternatively, or in addition, needle 1052 may take in a volume of aspacer gas, such as air 1080, between packets, while the needle is outof contact with liquid. The use of a spacer gas is optional. The spacerfluid may contain the same immiscible carrier fluid as the dropletpackets or a different immiscible carrier fluid. In some embodiments,the spacer fluid may be labeled, such as with a dye, to make itdistinguishable from the carrier fluid of a droplet packet and/or tomark a boundary (i.e., a leading or trailing end) of a droplet packet.Alternatively, or in addition, the spacer fluid and/or spacer region maybe distinguishable from a droplet packet by a decrease in concentration(i.e., an at least substantial absence) of droplets between dropletpackets.

J. Exemplary Systems with Multi-Stage Decoupling

FIGS. 17 and 18 show exemplary systems combining multi-stage decouplingof droplet generation from droplet loading into a reaction site, andalso show transport with autosampling.

FIG. 17 shows an example 1090 of system 850 of FIG. 10 that enablesmulti-stage decoupling of droplet generation and droplet loading into areaction site. More particularly, system 1090 provides storage of apacket of droplets first within an array of emulsions and then in adistinct storage site, after intake and prior to loading the packet intoa downstream reaction site. System 1090 may comprise an emulsion array1092 coupled to a drive assembly 1093. The emulsion array may be held bya plate 1094 (e.g., a microplate or droplet generator plate). System1090 also may comprise a droplet transporter 1096 that providesselectable intake, holding, heating, and loading.

Droplet transporter 1096 may incorporate an autosampler 1098, at leastone storage site 1100, and an outflow region 1102. Autosampler 1098 maytransfer droplet packets 1104-1108 into transporter 1096 from selectedwells of plate 1094, generally as described with respect to FIGS. 15 and16.

One or more valves 1110, 1112, in cooperation with one or more pumps1114, may be operated to determine the flow path and residency time ofeach packet. For example, valve 1110 may be operated to permit thedroplet packets to flow continuously to a downstream reaction site aftereach packet is transferred into transporter 1096. Alternatively, or inaddition, valve 1110 may be operated to transfer a droplet packet (ormultiple packets, see FIG. 16) along an inflow path, indicated by anarrow at 1116, to storage site 1100 (e.g., a holding channel or holdingchamber). Pump 1114 may be utilized to drive fluid movement into thestorage site.

Droplet packet 1106 may occupy storage site 1100 for any suitable periodof time. In some examples, packet 1106 may be heated by a heater 1118while the packet is disposed in the storage site. Alternatively, or inaddition, packet 1106 may be heated upstream of holding site 1100, suchas while the packet is contained by plate 1094, during flow to theholding site, and/or while disposed in outflow region 1102, amongothers. In any event, droplet packet 1106 may be permitted to leave theholding site by operation of valve 1110, to open an outflow path,indicated at 1120, to outflow region 1102. Also, pump 1114 may driveflow of droplet packet 1106 with the aid of a carrier fluid 1122obtained from a connected reservoir 1124. The carrier fluid also mayfunction to flush droplets from the holding site, to permit re-use ofthe site with a different packet of droplets without substantialcross-contamination. In any event, pump 1114 may drive packet 1106through outflow region 1102, and then another pump 1126 may drive thepacket to a downstream reaction site with the aid of a carrier fluid1128 obtained from a connected reservoir 1130. The use of downstreampump 1126 permits valve 1110 to be re-positioned, to close outflow path1120 and open inflow path 1116, such that pump 1114 can drive anotherpacket (e.g., packet 1104) into holding site 1100.

FIG. 18 shows another example 1140 of system 850 (see FIG. 10) thatenables multi-stage decoupling of droplet generation and droplet loadinginto a reaction site. System 1140 is related generally to system 1090 ofFIG. 17 but includes a plurality of isolatable storage sites 1142-1154that can be accessed in a selectable sequence, to provide loading ofdroplet packets from the storage sites into a reaction site according tothe sequence. System 1140 may comprise a serial arrangement of anemulsion array 1156 coupled to a drive assembly 1157. The emulsion arraymay be held by a plate 1158 (e.g., a droplet generator plate). System1140 also may comprise a droplet transporter 1160. The transporter mayenable selectable intake of droplet packets from plate 1158, holding ofeach packet for an adjustable period of time, and selectable loading ofthe packets into a reaction site.

Transporter 1160 may be equipped with an autosampler 1162, a temporaryholding station 1164, at least one pump 1166, and one or more valves1168-1172, among others. Pump 1166 may drive intake of droplets into anintake conduit 1174 of autosampler 1162. The droplets may represent onepacket or a plurality of spaced packets. In any event, pump 1166 maydrive flow of the packet into holding station 1164. Multi-position valve1170 then may be operated to open a flow path from holding station 1164to one of storage sites 1142-1154, and pump 1166 may drive the packetfrom the station to the storage site. This process may be repeated oneor more times to place other packets into other storage sites 1142-1154.A heater 1176 may apply heat to droplet packets disposed in the storagesites.

Droplet packets in the storage sites may be loaded serially into adownstream reaction site in a selectable order. In particularly, valve1170 may be positioned to open a flow path between a selected storagesite and station 1164. Pump 1166 then may drive a droplet packet(s) fromthe selected storage site into station 1164. Valve 1170 next may bere-positioned to open a flow path from station 1164 to an outflowconduit 1178. Then, pump 1166 may drive the droplet packet from station1164 to outflow conduit 1178, with the aid of a carrier fluid 1180traveling behind the packet. Pump 1166 may drive the packet from outflowconduit 1178 to a downstream reaction site, or another pump may beutilized (e.g., see FIG. 17). In some examples, the droplet packet(s) ina storage site may be driven to a waste reservoir 1182, instead of beingtransferred to station 1164.

K. Overview of Amplification in Static Fluid

FIGS. 19-21 relate to exemplary systems for sample analysis usingdroplet-based assays in which amplification is performed with stationaryemulsions and/or by batch amplification of an array of emulsions.

FIG. 19 shows a flowchart 1190 listing exemplary steps that may beperformed in a method of sample analysis using droplets subjected toconditions for amplification while disposed in a static fluid. The stepslisted may be performed in any suitable order and in any suitablecombination and may be combined with any other steps disclosed elsewhereherein.

A sample and at least one reagent may be mixed to create an assaymixture for amplification, indicated at 1192. The sample and reagent maybe combined manually or automatically. In some embodiments, one or moresamples and one or more reagents may be mixed to create a plurality ofdistinct and separate assay mixtures.

At least one emulsion may be generated from at least one assay mixture,indicated at 1194. The emulsion may be generated by serial, parallel, orbulk droplet generation (e.g., see Sections III and IV). If more thanone emulsion is generated, the emulsions may be generated in parallel orserially with respect to one another.

The at least one emulsion may be thermally cycled while the emulsionremains stationary, indicated at 1196. In particular, the emulsion maybe disposed in a container that restricts directional flow of theemulsion as it is thermally cycled.

Signals may be detected from droplets of the emulsion, indicated at1198. The signals may be detected while the emulsion is flowing or notflowing (e.g., see Section VI), and may involve serial droplet detectionor imaging, among others.

FIG. 20 shows a flowchart 1200 listing exemplary steps that may beperformed in a method of sample analysis using parallel amplification ofan array of emulsions. The steps listed may be performed in any suitableorder and in any suitable combination and may be combined with any othersteps disclosed elsewhere herein.

A plurality of assay mixtures may be created, indicated at 1202. Eachassay mixture may be an amplification mixture capable of amplifying atleast one species (or two or more species) of nucleic acid target, ifpresent, in the amplification mixture. The assay mixtures may containrespective distinct samples, distinct reagents (e.g., to amplifydifferent species of nucleic acid target), or any combination thereof.In some embodiments, the assay mixtures may be created or disposed in anarray, such as a planar array formed by a plate.

Emulsions may be generated from the respective assay mixtures, indicatedat 1204. The emulsions may be generated serially or in parallel withrespect to one another, and droplets of each emulsion may be generatedserially, in parallel, or in bulk.

The emulsions may be thermally cycled in an array, indicated at 1206.The array may be a linear array, a planar (two-dimensional) array, or athree-dimensional array.

Droplets signals may be detected from one or more droplets of eachemulsion, indicated at 1208. Detection may be performed while theemulsions remain disposed in the array and in a device holding theemulsions in the array (e.g., a plate). Alternatively, detection may beperformed after removal of droplets from the array. More particularly,detection may be performed after transfer of the droplets from acontainer/vessel (e.g., a plate, well, or a vial) that holds thedroplets. For example, the droplets may be transferred out of thecontainer/vessel to a detection site (e.g., a detection channel,chamber, recess) adjacent a detection window. Transfer may be achievedwith any suitable manual or automated fluid transfer device.Furthermore, detection may be flow-based detection (e.g., serial dropletdetection) or static/stopped-flow detection (e.g., imaging), amongothers.

FIG. 21 shows a schematic view of selected portions of an exemplarysystem 1210 for performing the method of FIG. 20. Any one component orcombination of the depicted system components may be omitted from thesystem, and any additional structural components disclosed elsewhereherein may be added to the system. The arrows indicate an exemplarysequence in which sample and emulsions may move between structuralcomponents of the system. However, the structural components may beutilized in a different sequence than shown here.

System 1210 may include a droplet generator array 1212, an emulsionholder 1214, a batch thermal cycler 1216, and a detector 1218. Dropletgenerator array 1212 may include a set of droplet generators connectedto one another in a linear, planar, or three-dimensional array.Alternatively, system 1210 may employ a plurality of droplet generatorsthat are not held in an array. In any event, a plurality of emulsionsmay be generated by the droplet generators and disposed in at least oneemulsion holder (e.g., a plurality of vials, or a plate with an array ofwells or chambers, among others). The emulsions may flow continuouslyfrom their respective droplet generators to the emulsion holder(s),which may be connected to the droplet generators. Alternatively, theemulsions may be transferred to the holder(s), such as with a manual orautomated fluid transfer device, at a selectable time. In any event, theemulsion holder(s) and the emulsions held therein may be thermallycycled by batch thermal cycler 1216 with the emulsions held in an array.Each site of the array may be defined by the emulsion holder, by areceiver structure of the thermal cycler, or both, among others. Afterthermal cycling, detector 1218 may be used to perform flow-based orstatic/stopped-flow detection of droplets. In some examples, thedetector may image droplets of the emulsions while the emulsions arestill disposed in the emulsion holder, and optionally, while theemulsion holder is operatively coupled to the thermal cycler.

L. Exemplary Droplet Generator Arrays for a Batch Amplification System

FIGS. 22-32 relate to exemplary devices for generating an array ofemulsions, which may (or may not) be reacted in parallel, such asbatch-amplified.

FIGS. 22 and 23 show an exemplary device 1220 equipped with an array ofdroplet generators. Device 1220 may be structured as a plateincorporating an array of droplet generators 1222. Each dropletgenerator may have any suitable droplet generator structure, such as anyof the structures described in Sections III and IV. Each dropletgenerator may include a plurality of reservoirs, such as wells 1224,1226, 1228 that can be accessed (e.g., fluid loaded and/or removed) fromabove the plate. The reservoirs may be termed ports and may be connectedfluidly by channels 1230 formed near the bottom of the reservoirs. Anintersection of the channels may form a site or intersection 1232 ofdroplet generation where droplets are formed by any suitable mechanism,such as flow-focusing.

FIG. 24 shows a schematic view of one of droplet generators 1222, whichhas a four-port configuration. To form droplets from the generator, oneor more oil wells 1224 may be loaded with a carrier fluid (e.g., oil).Also, a sample well 1226 may be loaded with a sample (e.g., an assaymixture, such as a PCR mixture including sample and reagent to perform areaction, such as amplification)). Pressure may be applied, indicated byvertical arrows at 1234, to oil wells 1224 and sample well 1226, todrive fluid flow, droplet generation, and flow of the resulting dropletsas an emulsion 1236 to emulsion well 1228. Fluid flow is indicated byarrows extending parallel to channels 1230. In other examples, eachdroplet generator may include only one oil well and one sample well, toprovide a three-port configuration (see below) or one or more oilreservoirs may be shared by droplet generators of the plate.

FIG. 25 shows a sectional view of plate 1220 assembled with an exemplarypressure manifold 1238 for applying pressure to droplet generators 1222(see FIGS. 22-24), to drive droplet generation (and emulsion formation).In this view, the wells are shown without fluid to simplify thepresentation. Also, the four wells visible in this view do not allbelong to the same droplet generator, but for simplification, thesewells are described as if they do.

Plate 1220 may include an upper member 1240 and a lower member 1242.Upper member 1240 may define wells 1224-1228, which may, for example, becreated by ridges 1244 (e.g., annular ridges; also see FIG. 23) thatproject upward from a base portion of the upper member and that formlaterally enclosing side walls of each well. The upper member also maydefine the top walls and side walls of channels 1230. These channels mayprovide communication for fluid movement from wells 1224, 1226 and towell 1228 of the droplet generator and may be formed in the bottomsurface of the upper member (such as in the cross pattern depicted inFIG. 23). Lower member 1242, which may be termed a cover layer, may bedisposed below upper member 1240 and attached to the upper member 1240via the bottom surface of the upper member. The lower member may overlapat least a portion of the upper member's bottom surface, from below, tocover and seal openings, such as channels 1230, formed in the bottomsurface of upper member 1240. Lower member 1242 thus may form a bottomwall of channels 1230, such that the channels are enclosed and fluidcannot escape from the bottom of the plate via the wells or thechannels. In some embodiments, upper member 1240 may be formed of apolymer, such as by injection molding.

Pressure manifold 1238 may include a manifold body or routing member1246 that is connected or connectable to one or more pressure sources1248, 1250. Manifold body 1246 may mate with plate 1220 from above toform a seal with wells 1224-1228 of the droplet generators via sealingelements or gaskets 1252, such as elastomeric O-rings. The manifold bodyalso may define channels 1254 that communicate with wells 1224-1228.

Any suitable combination of channels 1254 of the manifold body may beconnected or connectable to one or more pressure sources, to permitparallel or serial droplet generation from all or a subset of thedroplet generators. Accordingly, the pressure manifold may permitpressurization of only one of the droplet generators at a time, orparallel pressurization of two or more of the droplet generators, todrive parallel emulsion formation from two or more droplet generators ofthe plate in a batch process. For example, oil wells 1224 of a subset orall of the droplet generators may be pressurized with pressure source1250, and sample wells 1226 may be pressurized with another pressuresource 1248, to permit the pressures exerted on fluid in the oil wellsand the sample wells to be adjusted independently. Thus, in someexamples, the manifold may permit one pressure to be applied to the oilwells in parallel, and another pressure to be applied independently tothe sample wells in parallel. Alternatively, the same pressure sourcemay exert pressure on the oil wells and the sample wells. The manifoldfurther may permit emulsion wells 1228 to be independently pressurizedwith respect to the other wells (e.g., to form a pressure sink to drawfluid into the emulsion wells), may permit the emulsion wells to bevented during emulsion generation, indicated at 1256, to form a pressuredrop with respect to the pressurized oil and sample wells, or acombination thereof.

FIG. 26 shows plate 1220 with the pressure manifold replaced by anexemplary cover or sealing member 1258 after emulsion formation. (Anemulsion is present in emulsion well 1228, and the oil and assay mixturefluids are substantially depleted from wells 1224 and 1226.) Cover 1258may seal wells 1224-1228 to, for example, prevent fluid loss byevaporation. The cover may include a resilient member 1260 that engagesridges 1244 to cover and seal each well. In some examples, the resilientmember may be complementary to at least a portion of the wells, such asto form caps and/or plugs for individual wells. In some examples, cover1258 may cover and seal only emulsion wells 1228. In some examples, aplurality of covers may be used. In any event, after assembling plate1220 with cover 1258, the plate may be subjected to thermal cycling toinduce amplification in emulsion wells of the plate. For example, theplate and its cover may be disposed in a thermally cycled chamber.Alternatively, each emulsion may be transferred from plate 1220 toanother container, such as a sealable tube (e.g., for use with a CepheidSmartCycler) or a sealable well/chamber of a plate (e.g., a 96-well PCRplate), for thermal cycling. In other examples, sealing the emulsion ina container to reduce evaporation may not be required if the carrierfluid is capable of forming a sufficient liquid barrier to evaporationfor the droplets.

Droplet signals from the emulsions may be detected during/after thermalcycling, either with or without transfer of the emulsions from emulsionwells 1228 to a detection site. In some examples, plate 1220 may permitimaging from beneath the plate. In some embodiments, emulsion wells 1228may be sealed with a cover layer of optical quality (e.g., transparent),such as a tape or thin sheet, among others. The plate then may beinverted, and droplets imaged through the cover layer. In this case, thecarrier fluid and assay mixture compositions may be selected such thatthe droplets sink in the emulsion, to form a monolayer on the coverlayer. In some examples, the detector may be equipped with confocaloptics to enable collection of image data from droplets that are notdisposed in a monolayer.

Plate 1220 may have any suitable number of droplet generators 1222 (seeFIGS. 22-24), disposed in any suitable number of rows and columns. Insome embodiments, the droplet generators and/or wells thereof maycorrespond in spacing, number, and/or row/column arrangement to wells ofa standard microplate. For example, the center-to-center distance,number, and/or arrangement of droplet generators (and/or wells) maycorrespond to a microplate with 6, 24, 96, 384, 1536, etc. wells, amongothers. Thus, the plate may have 6, 24, 96, 384, or 1536 dropletgenerators and/or wells (total wells or of a given type (e.g., emulsionwells), which may be spaced by about 18, 9, 4.5, 2.25, or 1.125millimeters, among others. With an arrangement of ports corresponding toa standard microplate, instruments designed for parallel fluid transferto/from standard microplates may be utilized with plate 1220.

FIG. 27 shows another exemplary device 1270 incorporating an array ofdroplet generators 1272. Device 1270 may be structured as a plate andmay have any of the features described above for plate 1220 (see FIGS.22-26).

Each droplet generator 1272 may include a plurality of ports, which maybe structured as wells 1274-1278. In particular, droplet generator 1272may have a three-port configuration of an oil well 1274 to receive acarrier fluid, a sample well 1276 to receive a sample (e.g., a preparedsample that is an assay mixture, such as an amplification mixture), andan emulsion well 1278 to receive an overflow portion of an emulsiongenerated by the droplet generator.

FIG. 28 shows a bottom view of droplet generator 1272, taken aftergeneration of droplets 1280 to form an emulsion 1282. The dropletgenerator may include a network of channels 1284 that carry fluid fromoil well 1274 and sample well 1276 to a site or intersection 1286 ofdroplet generation. A pair of channels 1284 may extend from oil well1274 to site 1286 and another channel 1284 may extend from sample wellto site 1286, to form a cross structure at which droplets are formed byflow focusing of fluid from the sample well by carrier fluid disposed onopposing sides of fluid stream from the sample well.

Droplets 1280 may flow from droplet generation site 1286 to emulsionwell 1278 via an outlet channel 1288. The outlet channel may widen as itextends from site 1286 to form a chamber 1290. The chamber may have ahigh aspect ratio, with a height/thickness that generally corresponds tothe diameter of the droplets, to promote formation of a monolayer 1292of droplets in the chamber. Droplets also may flow past chamber 1290 toemulsion well 1278. However, emulsion well 1278 may functionpredominantly as an overflow site to collect excess emulsion. In otherembodiments, emulsion well 1278 may be omitted. In any event, chamber1290 may be connected to a vent 1294, which may be disposed generallydownstream of the chamber, to permit escape of air as an emulsion flowsinto the chamber.

FIG. 29 shows a sectional view of droplet generator 1272 and illustrateshow droplets may be generated and then imaged with an imager 1296 frombelow plate 1270. To generate droplets, oil well 1274 may be loaded witha carrier fluid 1298 and sample well 1276 with a sample (e.g., an assaymixture 1300). Pressure may be applied to the oil well and the samplewell, indicated by pressure arrows at 1302, to drive droplet generation.For example, pressure may be applied using a pressure manifold, asdescribed above for FIG. 25. In other examples, fluid flow and dropletgeneration may be driven by application of a vacuum to emulsion well1278, or by spinning plate 1270 in a centrifuge to apply a centripetalforce perpendicular to a plane defined by the plate, among others. Insome examples, plate 1270 may be designed with an oil reservoir thatsupplies carrier fluid to two or more droplet generators 1272. Inparticular, channels may extend from the oil reservoir to two or moresites 1286 of droplet generation. In other examples, pistons received inthe wells may be used to drive droplet generation (e.g., see SectionIII).

The droplets may be reacted in chamber 1290. For example, plate 1270 maybe placed in a heating station, such as a thermal cycler, to induceamplification of one or more nucleic acid targets in the droplets.Before heating the plate, wells 1274-1278 may be sealed from above withat least one sealing member, as described above for FIG. 26, to reduceevaporation. Alternatively, the plate may be heated without sealing thewells because fluid in the chamber may be resistant to evaporation.

Plate 1270 may be designed to permit imaging droplets in the chamber.For example, the plate may include an upper member 1304 attached to alower member 1306, as described above for plate 1220 (see FIGS. 25 and26), with at least one of the members forming a viewing window oroptical window 1308 through which the droplets may be imaged.Accordingly, the upper member and/or the lower member may betransparent, to permit imaging from above and/or below the plate. Plate1270 may provide the capability to image droplets in place, withoutunsealing any ports after reaction of the droplets (e.g., opening portsby removing a plate cover). Plate 1270 may reduce the risk of release ofamplicon formed in the plate during reaction, which could contaminateother subsequent reactions, because the amplicon can be held in the samesubstantially enclosed compartment (e.g., chamber 1290) during reactionand imaging. In some examples, the imaging device may be configured tocollected image data from droplets as they are being reacted, forexample, while they are being thermally cycled.

Chamber 1290 may have any suitable area. For example, the chamber mayhave a substantially larger footprint than a port, such as occupying atleast about 2, 5, or 10 times the area of the port.

FIG. 30 shows yet another exemplary device 1310 incorporating an arrayof droplet generators 1312. Device 1310 may be structured as a plate,and each droplet generator 1312 may be structured and may operategenerally as described above for droplet generators 1222 (see FIGS.22-26). In particular, each droplet generator may include a pair of oilwells 1314, a sample well 1316, and an emulsion well 1318.

FIG. 31 shows a bottom view of a droplet generator 1312 of plate 1310after droplet generation. The droplet generator may include a network ofchannels 1320 that permit flow of a carrier fluid and an assay mixture,respectively, from oil wells 1314 and sample well 1316 to a site 1322 ofdroplet generation. Droplets 1324 formed may flow into a chamber 1326 toform a substantial monolayer 1328 of droplets, as described above forchamber 1290 (see FIGS. 27-29).

FIG. 32 shows a sectional view of droplet generator 1312 and illustrateshow droplets may be generated and then imaged from below (and/or above)the device. In particular, plate 1310 may form a viewing window aboveand/or below chamber 1326.

M. Exemplary Detection for a Batch Amplification System

FIGS. 33-40 show exemplary modes of detection for a batch amplificationsystem.

FIG. 33 shows an exemplary imaging system 1360 for batch detection of anarray of emulsions 1362 that are held by a plate 1364 in an array ofwells 1366. The emulsions may be reacted (e.g., amplified by thermalcycling) in plate 1364 or may be transferred to the plate with a fluidtransfer device after reaction, among others. Plate 1364 may bedisposable (e.g., formed of plastic) or re-usable (e.g., formed ofquartz), depending on the application.

Imaging system 1360 may include an imaging device or imager 1368connected to a controller 1370, such as a computer. Any suitable aspectsof imaging system 1360 may be used in other imaging systems of thepresent disclosure. Also, imaging system 1360 may incorporate any otherfeature(s) disclosed for other imaging systems of the presentdisclosure. Imager 1368 may (or may not) be a fluorescence imager. Theimager may collect images of droplets disposed in wells 1366, forexample, using a CCD camera or a line-scan CCD, among others. For alarger field of view, plate 1364 and/or the camera may be placed on,and/or may be otherwise connected to, a translation stage to drivemotion in x-, y-, and, optionally, z-directions. In some examples,imager 1368 may, for example, include a laser/PMT device, as is used fordetection of microarrays. Further aspects of imaging devices and methodsthat may be suitable are described in Section VI.

FIG. 34 shows a fragmentary view of plate 1364, with well 1366 holdingan emulsion 1362 to be imaged. The well may include a bottom wall 1372,which may be flat, transparent, substantially non-fluorescent, or anycombination thereof, to make the well suitable for imaging from belowplate 1364. Well 1366 may have an inner surface that is hydrophobic,which may prevent aqueous droplets from wetting the well surface.

Well 1366 may contain a substantial monolayer 1374 of droplets 1376. Themonolayer may be disposed adjacent bottom wall 1372. Monolayer 1374 maybe obtained by selecting a suitable diameter of the well, number ofdroplets in the well, and size of each droplet. Also, monolayerformation may be promoted by selecting a carrier fluid composition thatis less dense than the fluid phase of the droplets, such that thedroplets sink to the bottom of the well. Monolayer formation also may bepromoted by spinning plate 1364 in a centrifuge.

FIGS. 35 and 36 show an exemplary imaging system 1380 for detectingimages of droplets held in one or more detection chambers, to provideparallel detection of droplets. System 1380 may include an imager 1382and at least one imaging slide 1384 operatively disposed with respect tothe imager, to permit image collection of droplets 1386 held by theslide.

Slide 1384 may define an imaging chamber 1388 and a viewing window 1390adjacent the imaging chamber. The imaging chamber may have a high aspectratio, with a length and width that are many times the height/thicknessof the chamber. Accordingly, imaging chamber 1388 may be sized to form amonolayer of droplets 1386 adjacent viewing window 1390, which may beformed by a bottom wall 1392 of the slide (see FIG. 36). In someexamples, the height of chamber 1388 may correspond to the diameter ofthe droplets, such as being about the same as the droplet diameter or nomore than about twice the droplet diameter, among others. The dropletsmay be loaded into the imaging slide (as part of an emulsion 1394) aftera reaction, such as amplification (e.g., thermal cycling), has beenperformed in the droplets. Alternatively, the emulsion may be loadedinto chamber 1388 before reaction, the slide optionally sealed, and thenthe emulsion reacted (e.g., thermally cycled) and imaged in the sameslide.

Imaging chamber 1388 may be connected to a pair of ports 1396, 1398,which may permit an emulsion to be introduced into and removed from thechamber (see FIG. 35). One or both of the ports may include a fitting1400 that enables sealed engagement with a flow-based fluid transferdevice 1402. The fluid transfer device, via either port, may introducefluid (e.g., an emulsion or wash fluid) into the chamber and may removeand/or flush fluid from the chamber (e.g., to permit the slide to bere-used and/or the emulsion to be collected). Slide 1384 may be imagedin any suitable orientation, such as horizontally, as shown in FIGS. 35and 36, vertically, or the like. Loading droplets into the imaging slidemay be performed with any suitable fluid transfer device (e.g., apipette, syringe, autosampler, etc.), which may be controlled (e.g.,positioned and actuated for fluid inflow and outflow) manually or with acontroller (e.g., a computer).

In other embodiments, droplet imaging may be performed with a slide thatlacks a chamber. For example, a cover slip may be utilized with theslide to form a monolayer of droplets between the slide and the coverslip. In this case, the slide may, for example, be a standard microscopeslide, a slide with a shallow well formed in one of its faces, a slidewith projections that space the cover slip from a planar surface of theslide, or the like.

Imaging system 1380 may be configured to image two or more slides 1384serially or in parallel. Accordingly, imager 1382 may have an imagingarea sufficient to encompass the viewing windows of two or more slidesat the same time. Alternatively, or in addition, imager 1382 may beoperatively coupled to a slide exchanger that can position a set ofslides serially in an imaging area of the imager, by adding each slideto the imaging area for imaging, and then removing the slide from theimaging area after imaging.

FIG. 37 shows an exploded view of an exemplary imaging system 1410including an imager 1412 and a vial 1414 that holds droplets 1416 to beimaged by the imager. Vial 1414 may define an inlet region or mouth 1418to receive the droplets from a fluid transfer device 1420, and animaging chamber 1422 to hold the droplets while they are imaged. Air maybe vented through the inlet region as an emulsion is loaded into thechamber or the vial may define a separate vent for this purpose. Chamber1422 may (or may not) have a high aspect ratio to promote formation of amonolayer of droplets. Also, the vial may include at least one viewingwindow 1424, which may be formed by one or more walls of the vial,through which light may be transmitted. The vial may be disposable(e.g., formed of a polymer) or re-usable (e.g., formed of quartz). Thevial may be spun in a centrifuge after loading and before imaging.Spinning may, for example, concentrate droplets in chamber 1422 and/orremove air bubbles from the detection chamber. Vial 1414 also mayinclude a cap 1426 to seal the vial. Droplets may be reacted (e.g.,amplified by thermal cycling) in the vial after loading and beforeimaging, or may be loaded after reaction. In other embodiments, the vialmay have any other suitable shape that defines a chamber, such as achamber including a planar surface, and forms a viewing window, such asa viewing window adjacent the planar surface.

FIG. 38 shows a schematic view of an exemplary system 1430 forstopped-flow imaging of reacted emulsions 1432 transported from anarray. Emulsions 1432 may be held in an array by a plate 1434 and may bereacted in the array or may be transferred to the array after reaction.The emulsions (or at least a portion thereof) may be transportedserially to at least one imaging chamber 1436 using an autosampler 1438connected to an injection valve 1440. Exemplary imaging chambers thatmay be suitable are shown in FIGS. 35 and 36 of this Section and inSection VI. The injection valve may be used to control filling, holding,emptying, and, optionally, flushing the imaging chamber. An imager 1442may be operatively disposed with respect to a viewing window 1444adjacent the imaging chamber, to provide image collection of dropletsdisposed in the imaging chamber. After each emulsion is imaged, theemulsion may be removed from the imaging chamber by flow to awaste/collection reservoir 1446. Further aspects of autosamplers aredescribed above in relation to FIGS. 15-18.

FIG. 39 shows a schematic view of another exemplary system 1450 forstopped-flow imaging of reacted emulsions transported from an array.System 1450 is related to system 1430 of FIG. 38 but includes aplurality of imaging chambers 1452. One or more inlet valves 1454 and/oroutlet valves 1456 may be operated to determine an order in which theimaging chambers are filled with emulsions, isolated from fluid flow forimaging, emptied, and/or flushed, among others.

FIG. 40 shows a schematic view of an exemplary system 1460 for transportof reacted emulsions 1462 from an array to a detection channel 1464, forserial droplet detection. System may include an autosampler 1466 and aninjection valve 1468 that serially load emulsions 1462 into detectionchannel 1464, for flow past a viewing window 1470 that is operativelydisposed with respect to a detector 1470. A flow-focusing assembly 1472may focus droplets in the flow stream before they reach detectionchannel 1464. Further aspects of flow-focusing upstream of a detectionchannel are described in Section VI.

N. Additional Embodiments

This example describes additional aspects of system architecture, inaccordance with aspects of the present disclosure, presented withoutlimitation as a series of numbered sentences.

(i). Flow System

1. A system for analyzing a sample, comprising (A) a droplet generatorconfigured to generate droplets containing portions of a sample to beanalyzed, the droplets being disposed in an immiscible fluid forming asample emulsion, (B) a heating and cooling station having a fluid inletand a fluid outlet, (C) a detection station downstream from the heatingand cooling station, (D) a channel forming a single-pass continuousfluid route from the fluid inlet to the fluid outlet of the heating andcooling station, (E) a pump for moving the sample emulsion through thechannel, (F) a controller programmed to operate fluid transport throughthe channel, and (G) an analyzer configured to process data collected atthe detection station.

2. The system of paragraph 1, wherein the detection system is situatedto detect presence of target in the sample emulsion after passingthrough the heating and cooling system.

3. The system of paragraph 1 further comprising a droplet reservoir, afirst fluid conduit connecting the droplet generator to the reservoir,and a second fluid conduit connecting the reservoir to the fluid inletof the heating and cooling station.

4. The system of paragraph 1, wherein the droplet generator is adaptedfor single-use detachable connection to the heating and cooling stationwithout exposing the heating and cooling station to contamination fromsample contained in the sample emulsion.

5. The system of paragraph 1, wherein the droplet generator isconfigured to generate the sample emulsion external to the heating andcooling station.

6. The system of paragraph 1, wherein the heating and cooling stationincludes multiple heating zones along the fluid route configured forperforming a polymerase chain reaction on a nucleic acid targetcontained in a droplet.

7. The system of paragraph 1, wherein the heating and cooling stationincludes at least one thermoelectric cooler.

8. The system of paragraph 1, wherein the controller is programmed toadjust the droplet generator to alter droplet size based on datareceived from the detection station.

9. The system of paragraph 1, wherein the controller is programmed toalter sample concentration prior to droplet generation based on datareceived from the detection station.

10. The system of paragraph 1, wherein the controller is programmed toalter a sample preparation procedure prior to droplet generation in thedroplet generator based on data received from the detection station.

11. The system of paragraph 1, wherein the analyzer is programmed todetermine a concentration of a target molecule in the sample based atleast partially on the frequency of droplets containing the target outof a population of droplets containing sample portions.

12. The system of paragraph 1, wherein the droplet generator includes asample reservoir, an oil source, an oil/sample intersection, and anemulsion outlet, the emulsion outlet having a distal end portion adaptedfor detachable sealed engagement with a receiving port on the heatingand cooling station.

13. The system of paragraph 1, wherein the droplet generator iscontained in a cartridge having at least one piston for drivingemulsification.

14. The system of paragraph 1, wherein the droplet generator iscontained in a cartridge having at least one piston for pumping sampleemulsion through the channel network.

15. The system of paragraph 1, wherein the channel includes a helicalcapillary tube portion passing through the heating and cooling station.

16. The system of paragraph 15, wherein the capillary tube portion has adiameter approximately equal to the diameter of droplets generated bythe droplet generator.

17. The system of paragraph 1, wherein the capillary tube portionincludes a hot-start segment passing through a hot-start zone prior to adenaturation zone in the heating and cooling station.

18. The system of paragraph 1, wherein the heating and cooling stationincludes thermoelectric coolers configured for controlling temperaturesin heating and cooling zones by transferring heat between a thermal coreand the heating and cooling zones.

19. The system of paragraph 15, wherein the helical capillary tubeportion defines a helical path that decreases in length over successivecycles.

20. The system of paragraph 1, wherein the heating and cooling stationincludes (a) a core defining a central longitudinal axis, (b) aplurality of segments attached to the core and defining a plurality oftemperature regions; and (c) a plurality of heating elements configuredto maintain each temperature region approximately at a desiredtemperature, a portion of the channel configured to transport a sampleemulsion cyclically through the temperature regions.

21. The system of paragraph 20, wherein the plurality of segmentsincludes a plurality of inner segments defining the plurality oftemperature regions and a plurality of outer segments attached to theinner segments, and wherein the portion of the channel is disposedbetween the inner and outer segments.

22. The system of paragraph 21, wherein the portion of channel includesfluidic tubing that wraps around the inner segments.

23. The system of paragraph 21, wherein the fluidic tubing is disposedin grooves of the inner segments that wrap substantially helicallyaround the inner segments.

24. The system of paragraph 1, wherein the droplet generator iscontained in a disposable cartridge.

25. The system of paragraph 24, wherein the cartridge includes a celllysing region, a separating region, a reagent mixing region, and adroplet generation region for extracting nucleic acid from a sample andformation of droplets into a heat stable sample emulsion.

26. The system of paragraph 1, wherein the channel has open ends forpermitting continuous flow of a sample emulsion.

27. The system of paragraph 1, wherein the droplet generator is capableof generating a heat stable sample emulsion.

(ii). Droplet Generator Plate

1. A device for generating an array of emulsions, comprising a plateincluding one or more oil reservoirs and forming an array of emulsiongenerator units, each unit including a sample port, a droplet collectionsite, and a channel intersection that receives a sample from the sampleport and a carrier fluid from at least one oil reservoir and generatesan emulsion of sample droplets in the carrier fluid that flows to thedroplet collection site.

2. The device of paragraph 1, wherein the sample port is a well thatpermits sample loading from above the plate.

3. The device of paragraph 1, wherein each emulsion generator unitincludes at least one oil reservoir.

4. The device of paragraph 3, wherein the at least one oil reservoir isa well that permits loading of the carrier fluid from above the plate.

5. The device of paragraph 1, wherein the sample ports collectively forma port array, and wherein the port array is arranged in correspondencewith wells of a standard microplate.

6. The device of paragraph 5, wherein the plate has 96 sample ports.

7. The device of paragraph 1, wherein the channel intersection includesa pair of oil inlets, and wherein the pair of oil inlets connect to oneor more oil reservoirs.

8. The device of paragraph 7, wherein channel intersection includes asample inlet that receives sample from the sample port, and wherein thepair of oil inlets flank the sample inlet on opposing sides of thesample inlet.

9. The device of paragraph 1, wherein the droplet collection siteincludes a well.

10. The device of paragraph 1, wherein the droplet collection sitedefines a cavity bounded by walls of the plate disposed above and belowthe cavity.

11. The device of paragraph 10, wherein the cavity has a height thatcorresponds in size to the droplets such that a substantial monolayer ofthe droplets is formed in the cavity when the emulsion flows into thecavity.

12. The device of paragraph 10, wherein the cavity has a width and athickness, and wherein the width is at least about ten times thethickness.

13. The device of paragraph 10, wherein an outlet channel extends fromthe channel intersection to the droplet collection site, wherein theplate defines a plane, and wherein the cavity and the outlet channeleach have a width measured parallel to the plane, and wherein the widthof the cavity is substantially greater than the width of the outletchannel.

14. The device of paragraph 10, wherein the cavity is a chamber, andwherein the chamber is connected to a vent that permits escape of gasfrom the chamber as the emulsion flows into the chamber.

15. The device of paragraph 1, wherein the droplet collection sitedefines a cavity and includes a window formed by a transparent wall ofthe plate adjacent to the cavity, and wherein the window permits opticaldetection, through the transparent wall, of droplets in the cavity.

16. The device of paragraph 15, wherein the window is formed below thecavity.

17. The device of paragraph 1, wherein the plate includes an uppermember attached to a lower member, wherein the upper member defines thesample port, wherein an upper region of the channel intersection isformed in a bottom surface of the upper member, and wherein the lowermember is attached to the bottom surface to form a bottom wall of thechannel intersection.

18. The device of paragraph 1, further comprising a cover that assembleswith the plate to seal the sample ports.

19. The device of paragraph 1, wherein the emulsion generator units arearranged in rows and columns with two or more units per row and percolumn.

(iii). Batch Array Method

1. A method of sample analysis, comprising (A) forming an array ofemulsions, each emulsion including partitions of a respective sampledisposed in droplets; (B) applying heat to the emulsions while they aredisposed in the array, to induce nucleic acid amplification in dropletsof the emulsions; (C) detecting signals from droplets of each emulsion;and (D) estimating a presence, if any, of a nucleic acid target in eachrespective sample based on the signals detected.

2. The method of paragraph 1, wherein the step of forming includes astep of generating the emulsions with a plate that includes an array ofemulsion generator units.

3. The method of paragraph 2, wherein the plate includes a plurality ofreservoirs to hold the respective samples, and wherein the step ofgenerating includes a step of applying pressure to the plurality ofreservoirs after placing the respective samples into the reservoirs.

4. The method of paragraph 2, wherein the step of generating includes astep of spinning the plate in a centrifuge.

5. The method of paragraph 2, wherein the step of forming includes astep of removing each emulsion from the plate and disposing suchemulsion at a position within the array.

6. The method of paragraph 2, wherein the plate defines an array ofsample ports that open upwardly, and wherein the step of generatingincludes a step of disposing each respective sample in a sample port.

7. The method of paragraph 2, wherein the step of applying heat isperformed with the emulsions held in the array by the plate.

8. The method of paragraph 1, wherein the step of applying heat isperformed with the emulsion disposed in a cavity, wherein the cavity hasa width and a thickness, and wherein the width is many times thethickness.

9. The method of paragraph 8, wherein the width is at least about tentimes the thickness.

10. The method of paragraph 1, wherein the step of applying heatincludes a step of heating the emulsions to a temperature sufficient tomelt nucleic acid duplexes in the droplets.

11. The method of paragraph 1, wherein the step of applying heatincludes a step of thermally cycling the array of emulsions to induceamplification by PCR.

12. The method of paragraph 1, wherein the step of detecting signalsincludes a step of imaging droplets of each emulsion.

13. The method of paragraph 12, wherein the step of imaging droplets isperformed while the emulsions are still disposed in the array.

14. The method of paragraph 13, wherein the step of forming includes (a)a step of generating droplets of each emulsion with a plate and (b) astep of collecting the emulsions in an array of chambers defined by theplate, wherein the step of applying heat is performed while theemulsions are disposed in the array of chambers, and wherein the step ofimaging is performed through a transparent window formed by a wall ofthe plate adjacent to each chamber.

15. The method of paragraph 11, wherein the step of thermally cycling isperformed without sealing the plate from above after disposing theemulsions in the array of chambers.

16. The method of paragraph 1, further comprising a step of transferringat least a portion of each emulsion out of the array and to a detectionstation after the step of applying heat.

17. The method of paragraph 16, wherein the step of transferring isperformed serially with the emulsions.

18. The method of paragraph 16, wherein the step of transferring isperformed with an autosampler.

19. The method of paragraph 16, wherein the step of detecting signalsincludes a step of detecting droplet signals serially as droplets flowpast a detection window.

20. The method of paragraph 16, wherein the step of detecting includes astep of imaging droplets.

21. The method of paragraph 1, wherein the step of estimating a presenceprovides a qualitative determination of whether the nucleic acid targetis present or absent in the respective sample.

22. The method of paragraph 1, wherein the step of estimating a presenceincludes a step of estimating a concentration and/or a copy number ofthe nucleic acid target in the respective sample.

23. The method of paragraph 22, wherein the step of estimating apresence includes a step of assigning a starting copy number of two ormore molecules of a nucleic acid target to at least one of the dropletsbased on one or more detected signals.

24. The method of paragraph 1, wherein the step of estimating includes astep of utilizing an algorithm based on Poisson statistics.

25. The method of paragraph 1, wherein the step of applying heat inducesnucleic acid amplification of respective different species of nucleicacid target in at least two of the emulsions.

26. The method of paragraph 1, wherein the step of applying heat inducesnucleic acid amplification of two or more distinct species of nucleicacid target in at least one of the emulsions, and wherein the step ofestimating includes a step of estimating a presence for each of thedistinct species of nucleic acid target.

(iv). Single Emulsion—Batch Amplification

1. A method of sample analysis, comprising (A) forming an emulsionincluding droplets disposed in a carrier fluid, each droplet containinga partition of a sample prepared as a reaction mixture for amplificationof a nucleic acid target; (B) disposing at least a portion of theemulsion in a chamber that is many times wider than an average diameterof the droplets; (C) applying heat to the at least a portion of theemulsion disposed in the chamber to induce nucleic acid amplification indroplets; (D) detecting signals from droplets of the emulsion; and (E)estimating a presence, if any, of the nucleic acid target in the samplebased on the signals detected.

2. The method of paragraph 1, wherein the emulsion flows continuouslyinto the chamber from a site of droplet generation.

3. The method of paragraph 1, wherein the step of applying heat includesa step of thermal cycling the at least a portion of the emulsion toinduce PCR amplification of the nucleic acid target.

4. The method of paragraph 1, wherein the chamber is at least about tentimes wider than the average diameter of the droplets.

5. The method of paragraph 1, wherein the step of detecting signalsincludes a step of collecting an image of a plurality of the droplets.

6. The method of paragraph 1, wherein the step of detecting signalsincludes a step of detecting signals serially from the droplets as suchdroplets are traveling through a detection station.

7. The method of paragraph 1, wherein the droplets form a substantialmonolayer in the chamber.

8. The method of paragraph 7, wherein the average separation betweenadjacent pairs of droplets in the chamber is less than an averagediameter of the droplets.

(v). System for Batch Amplification

1. A system for sample analysis, comprising (A) a droplet generator thatforms an emulsion including droplets that each contain a partition of asample prepared as a reaction mixture for amplification of a nucleicacid target; (B) an emulsion holder defining a cavity to contain atleast a portion of the emulsion, the cavity being many times wider thanan average diameter of the droplets; (C) a heating station to apply heatto the at least a portion of the emulsion disposed in the cavity toinduce nucleic acid amplification in droplets; (D) a detection stationto detect signals from droplets of the emulsion; and (E) a controller incommunication with the detection station and programmed to estimate apresence, if any, of the nucleic acid target in the sample based on thesignals detected.

2. The system of paragraph 1, further comprising a plate including thedroplet generator and a plurality of other droplet generators.

3. The system of paragraph 1, wherein the emulsion holder is connectedto the droplet generator such that generated droplets flow continuouslyinto the cavity.

4. The system of paragraph 1, wherein the detection station includes atleast one detection chamber and at least one imaging device to collectimages of droplets disposed in the detection chamber.

5. The system of paragraph 1, further comprising a fluid transfer deviceto transfer droplets from the cavity to the detection station.

6. The system of paragraph 1, wherein the fluid transfer device is amanually controlled pipette.

7. The system of paragraph 1, wherein the fluid transfer device is anautosampler.

8. The system of paragraph 1, wherein the cavity has a thickness thatcorresponds to the average diameter of the droplets such that thedroplets form a substantial monolayer in the cavity.

9. The system of paragraph 1, wherein the cavity is a chamber.

10. The system of paragraph 1, wherein the cavity is at least ten timeswider than the average diameter of the droplets.

(vi). High Throughput System

1. A system for droplet-based sample analysis, comprising (A) a sampleinput station to hold a plurality of emulsions each including partitionsof a respective sample disposed in droplets; (B) a heating station toapply heat to droplets to induce amplification of a nucleic acid target,if present, in individual droplets; (C) a detection station to detectsignals from droplets that have been heated by the heating station; (D)a fluidics network connecting the sample input station, the heatingstation, and the detection station, to provide fluid flow from thesample input station to the heating station and the detection station;and (E) a controller programmed to control an order in which packets ofdroplets from the emulsions are transferred from the sample inputstation to the heating station, and to estimate a presence of a nucleicacid target in samples corresponding to the packets based on signalsfrom the detection station.

2. The system of paragraph 1, wherein the fluidics network includes aholding station to store packets of droplets upstream from the heatingstation.

3. The system of paragraph 2, wherein the controller is programmed tocontrol a sequence in which packets are transferred into the holdingstation from the sample input station and also to control a sequence inwhich such packets are loaded into the heating station from the holdingstation.

4. The system of paragraph 3, wherein at least a portion of at least oneof the sequences is selected by the controller based on signals detectedby the detection station.

5. The system of paragraph 2, wherein the holding station includes aplurality of discrete storage sites, and wherein the controller isprogrammed to control loading of packets into the storage sites andunloading of the packets from the storage sites.

6. The system of paragraph 5, wherein holding station is designed topermit loading the storage sites with packets in an arbitrary order andunloading the packets from the storage sites in an arbitrary order.

7. The system of paragraph 2, wherein the holding station includes atleast one heater configured to apply heat to packets disposed in theholding station.

8. The system of paragraph 1, wherein the controller is programmed tocontrol formation of a spacer segment of fluid in the fluidics networkbetween adjacent packets of droplets as the adjacent packets areintroduced into the fluidics network from the sample input station.

9. The system of paragraph 1, wherein the fluidics network includes anautosampler that picks up packets of droplets from the sample inputregion and loads such packets into the heating station.

10. The system of paragraph 1, wherein the controller is programmed toreceive inputs from a user selecting a sequence and to control transferof packets to the heating station according to the sequence.

11. The system of paragraph 1, wherein the detection station detectssignals from droplets disposed in a flow stream.

11. The system of paragraph 1, wherein the detection station collectsimages of droplets.

12. The system of paragraph 1, wherein the detection station detectsfluorescence signals from droplets.

(vii). Batch System I

1. A system for sample analysis, comprising (A) at least one dropletgenerator that forms a plurality of emulsions including droplets thateach contain a sample partition prepared as a reaction mixture foramplification of a nucleic acid target; (B) a plate defining an array ofcavities to hold the emulsions; (C) a heating and cooling device to heatthe emulsions disposed in the cavities to induce nucleic acidamplification in droplets; (D) a detection assembly to detect signalsfrom intact droplets of the emulsions; and (E) a controller incommunication with the detection assembly and programmed to estimate apresence, if any, of the nucleic acid target in a sample based onsignals detected from the intact droplets.

2. The system of paragraph 1, wherein the droplet generator isintegrated with the plate.

3. The system of paragraph 2, wherein each cavity is supplied by aseparate droplet generator.

4. The system of paragraph 2, wherein each cavity is supplied by thesame droplet generator.

5. The system of paragraph 1, wherein the droplet generator is not partof the plate.

6. The system of paragraph 1, wherein the droplet generator includes atleast one oil reservoir, a sample reservoir, and a fluid path from eachreservoir to at least one cavity.

7. The system of paragraph 1, further comprising a pressure source thatdrives droplet generation.

8. The system of paragraph 1, wherein the detection assembly isconfigured to detect signals from droplets while disposed in thecavities.

9. The system of paragraph 1, further comprising a fluid transfer deviceconfigured to transfer droplets from the cavities to a detection site ofthe detection assembly.

10. The system of paragraph 9, wherein the detection site is separatefrom the plate.

11. The system of paragraph 9, wherein the detection assembly isconfigured to detect droplets serially.

12. The system of paragraph 9, wherein the detection assembly isconfigured to image batches of droplets.

13. The system of paragraph 12, wherein the detection assembly isconfigured to image droplet batches serially, each droplet batchcorresponding to a different emulsion.

14. The system of paragraph 1, wherein the detection assembly includesconfocal optics.

15. The system of paragraph 1, wherein each cavity is bounded above andbelow by walls of the plate.

16. The system of paragraph 1, wherein each cavity is bounded by atransparent wall of the plate that permits detection of droplets in suchcavity through the transparent wall.

17. The system of paragraph 1, wherein the droplet generator includes asample reservoir that opens upwardly to permit sample loading from abovethe plate.

18. The system of paragraph 1, wherein the cavity is a well, furthercomprising a sealing member to seal the well.

19. The system of paragraph 1, wherein the droplet generator includesone or more orifices from which the droplets are generated serially.

20. The system of paragraph 1, wherein the droplet generator isconfigured to form droplets that are monodisperse.

21. The system of paragraph 1, wherein the controller is configured toestimate the presence of the nucleic acid target based on a percentageof droplets that are determined to be positive for amplification of thenucleic acid target.

(viii). Batch System II

1. A system for sample analysis, comprising (A) a droplet generatorincluding an oil reservoir, a sample reservoir, a cavity, and a channelintersection that receives a sample from the sample reservoir and acarrier fluid from the oil reservoir and generates droplets that flow tothe cavity as an emulsion; and (B) a heating device to heat the dropletgenerator to induce nucleic acid amplification in droplets of theemulsion in the cavity.

2. The system of paragraph 1, further comprising a plate that includesthe droplet generator and a plurality of other droplet generators.

3. The system of paragraph 1, further comprising a pressure source thatdrives droplet generation.

4. The system of paragraph 3, wherein the pressure source includes amanifold that forms a sealed relation with the droplet generator.

5. The system of paragraph 1, further comprising a detection assembly todetect signals from droplets of the emulsion.

6. The system of paragraph 5, wherein the detection assembly isconfigured to detect signals from droplets while the droplets aredisposed in the cavity.

7. The system of paragraph 5, wherein the detection assembly isconfigured to detect signals from the droplets while the dropletgenerator is thermally coupled to the heating device.

8. The system of paragraph 5, wherein the detection assembly isconfigured to image a batch of droplets.

9. The system of paragraph 8, wherein the detection assembly includesconfocal optics.

10. The system of paragraph 5, further comprising a controller incommunication with the detection assembly and programmed to estimate apresence, if any, of a nucleic acid target in the sample based on thesignals detected.

11. The system of paragraph 1, wherein the heating device includes atemperature-controlled chamber that receives the droplet generator.

12. The system of paragraph 1, wherein the heating device is a heatingand cooling device that thermally cycles the droplet generator to inducePCR amplification in the droplets of the emulsion in the cavity.

13. The system of paragraph 1, wherein the cavity is bounded above andbelow by walls of the droplet generator.

14. The system of paragraph 1, wherein the cavity is bounded by atransparent wall of the droplet generator that permits detection ofdroplets in the cavity through the transparent wall.

15. The system of paragraph 1, wherein the cavity is a well, furthercomprising a sealing member to seal the well.

(ix). Miscellaneous 1

1. A method of sample analysis, comprising (A) generating a plurality ofdroplets from a sample, each droplet containing a mixture to testoccurrence of a reaction; (B) storing a packet of the droplets for aselectable time period; (C) introducing at least a portion of the packetinto a channel after the step of storing; (D) subjecting the portion ofthe packet to one more conditions that promote occurrence of thereaction by moving the at least a portion of the packet along thechannel; and (E) performing, after the step of subjecting and on each ofa plurality of droplets of the at least a portion of the packet, atleast one measurement related to occurrence of the reaction.

2. The method of paragraph 1, wherein the step of generating includes astep of generating the plurality of droplets by fluid flow from at leastone orifice.

3. The method of paragraph 1, wherein the step of generating includes astep of generating droplets with each droplet capable of amplificationof a nucleic acid target, if present, in the droplet, wherein the stepof subjecting includes a step of subjecting the at least a portion ofthe packet to conditions that promote amplification of the nucleic acidtarget in droplets of the at least a portion of the packet, and whereinthe step of performing includes a step of performing the at least onemeasurement to permit determination of whether amplification of thenucleic acid target occurred in individual droplets.

4. The method of paragraph 1, wherein the step of storing includes astep of storing the packet of droplets in a compartment that is in fluidisolation from the channel, and wherein the step of introducing includesa step of placing the compartment and the channel in fluid communicationwith one another.

5. The method of paragraph 1, wherein the packet of droplets is disposedin a volume of carrier fluid, wherein the step of storing includes astep of stopping flow of the volume of carrier fluid, and wherein thestep of introducing includes a step of starting flow of at least aportion of the volume of carrier fluid.

6. The method of paragraph 1, wherein the step of subjecting includes astep of thermally cycling the at least a portion of the packet.

7. The method of paragraph 1, further comprising (1) a step ofdetermining a number of droplets in which amplification of a nucleicacid target occurred based on data obtained from the step of performing,and (2) a step of estimating a total presence of the nucleic acid targetin the sample based on the number of droplets.

8. The method of paragraph 1, wherein the steps of storing, introducing,subjecting, and performing are performed with a plurality of differentpackets, and wherein the packets are introduced serially into thechannel.

9. The method of paragraph 8, further a step of selecting a relativeorder in which at least two of the different packets are introduced intothe channel.

10. The method of paragraph 9, wherein the step of selecting is based ona result obtained based on the step of performing with droplets ofanother packet.

11. A method of sample analysis for a nucleic acid target, comprising(A) generating a plurality of droplets from a sample, each droplet beingcapable of amplification of a nucleic acid target, if present, in thedroplet; (B) storing a packet of the droplets for a selectable timeperiod; (C) introducing at least a portion of the stored packet into achannel; (D) moving the portion of the packet along the channel suchthat the portion is subjected to conditions that promote amplificationof the nucleic acid target in droplets of the portion; and (E)performing at least one measurement related to amplification of thenucleic acid target on each of a plurality of droplets after the step ofmoving.

12. A method of sample analysis, comprising (A) providing a channel, anarray of samples, an array of reagents, and predefined flow pathsconnecting all of the samples and reagents to the channel, to permitselection of any combination of sample and reagent from the arrays; (B)selecting a combination of a sample from the array of samples and areagent from the array of reagents; (C) generating droplets eachincluding the combination and containing an assay mixture to be testedfor occurrence of a reaction involving the sample and the reagentselected; (D) introducing a plurality of the droplets into the channel;(E) subjecting the plurality of droplets to one or more conditions thatpromote occurrence of the reaction while moving the plurality ofdroplets along the channel; and (F) performing at least one measurementrelated to occurrence of the reaction on one or more of the plurality ofdroplets after the step of subjecting.

14. The method of paragraph 12, wherein the combination is a firstcombination, further comprising a step of selecting a second combinationof sample and reagent from the arrays, wherein the steps of generating,introducing, subjecting, and performing are repeated with the secondcombination.

15. The method of paragraph 14, wherein the second combination isselected based on a result obtained using data from the step ofperforming at least measurement on the first combination.

16. The method of paragraph 14, further comprising a step of changingthe array of samples to add or subtract at least one sample, the arrayof reagents to add or subtract at least one reagent, or both, andwherein the step of selecting a second combination selects a combinationafter the step of changing.

17. The method of paragraph 16, wherein the step of changing isperformed while the step of subjecting is performed with the firstcombination.

18. The method of paragraph 14, wherein the step of selecting a secondcombination of sample and reagent is performed based on a user commandreceived after the step of selecting a first combination.

19. The method of paragraph 18, wherein the user command is receivedduring the step of subjecting with the first combination.

20. The method of paragraph 19, wherein the step of introducing for thefirst combination is performed until a predefined condition is satisfiedif the user command is not received, and wherein the step of introducingis interrupted by the user command before the predefined condition issatisfied.

21. The method of paragraph 20, wherein the predefined condition is apredefined number of droplets introduced, a predefined time intervalduring which droplets are introduced, or both.

22. The method of paragraph 14, wherein the array of reagents includesdifferent pairs of primers for amplification of different nucleic acidtargets.

23. A method of sample analysis, comprising (A) providing a channel, anarray of samples, an array of reagents, and predefined flow pathsconnecting all of the samples and reagents to the channel; (B) selectingfirst and second combinations of sample and reagent from the arrays; (C)generating a first packet of droplets each including the firstcombination and a second packet of droplets each including the secondcombination; (D) introducing a plurality of droplets of the first packetand of the second packet serially into the channel; (E) subjecting theplurality of droplets of each packet to one or more conditions thatpromote occurrence of a reaction involving the first combination or thesecond combination while moving each plurality of droplets along thechannel; and (F) performing at least one measurement related tooccurrence of the reaction on one or more of the plurality of dropletsafter the step of subjecting.

24. An apparatus for sample analysis, comprising (A) an adjustablenumber of ports to receive samples; (B) an adjustable number of sites tohold reagents; (C) a channel that extends through one or moretemperature-controlled zones and that connects to the ports and thesites by predefined flow paths; (D) a droplet generator that generatesdroplets of a selected combination of a sample and a reagent forintroduction into the channel; (E) a detector positioned to provide oneor more measurements on droplets of the selected combination after thedroplets have been disposed in at least one temperature-controlled zone;and (F) a controller that controls combination of samples with reagents.

(x). Miscellaneous 2

1. A system for generating microdroplets comprising (A) asample-containing apparatus comprising a sample containing chamber and afirst microfluidic channel having an inlet end and an outlet end,wherein the inlet end of the first microfluidic channel is connected tothe sample containing chamber; and (B) a microdroplet generatorapparatus comprising the outlet end of the first microfluidic channel, asecond microfluidic channel having an inlet end, and a spacer regionthat is filled with an immiscible fluid, wherein the outlet end of thefirst microfluidic channel forms one wall of the microdroplet generatorapparatus, the inlet end of the second microfluidic channel formsanother wall of the microdroplet generator region, and the spacer regionseparates the first microfluidic channel outlet end from the secondmicrofluidic channel inlet end such that the first microfluidic channeloutlet end only contacts the immiscible fluid.

2. The system of paragraph 1, wherein the sample containing apparatus isremovable.

3. The system of paragraph 1, wherein the immiscible fluid is an oil.

4. A method of nucleic acid amplification comprising (A) diluting orconcentrating a sample comprising a plurality of nucleic acid targetsand components for performing nucleic acid amplification; (B) producingmicrodroplets within an immiscible fluid in a capillary tube, wherein aplurality of microdroplets containing a single nucleic acid templatefrom the plurality of nucleic acid targets is formed, and wherein thetube has a first open end for fluid inlet and a second open end forfluid outlet to permit a continuous flow; and (C) amplifying the singlenucleic acid template in the microdroplets by heating and cooling suchthat a plurality of single nucleic acid templates within themicrodroplets are amplified.

5. The method of paragraph 4, wherein the microdroplets comprise atleast 2 different size microdroplets.

6. The method of paragraph 4, wherein a first microdroplet size isbetween 20 and 100 microns, and a second microdroplet size is between100 and 250 microns.

7. A method of nucleic acid amplification of a sample, comprising (A)providing a biological sample; (B) producing microdroplets within animmiscible fluid in a capillary tube, wherein the microdroplets comprisenucleic acids and components for performing nucleic acid amplificationand wherein the tube has a first open end for fluid inlet and a secondopen end for fluid outlet to permit a continuous flow and the tube is incontact with at least two solid heating blocks, wherein the heatingblocks are maintained at different temperatures and the temperature ofat least one heating block is controlled by a thermoelectric controller;(C) moving the microdroplets through the tube; and (D) thermally cyclingthe microdroplets in the tube to amplify the nucleic acids.

8. A sequence detection system able to detect a single nucleic acidmutation using the method of (A) producing microdroplets within animmiscible fluid in a capillary tube, wherein a plurality ofmicrodroplets containing a single nucleic acid template from theplurality of nucleic acid targets is formed; (B) amplifying the singlenucleic acid template in the microdroplets by heating and cooling suchthat a plurality of single nucleic acid templates within themicrodroplets are amplified; and (C) detecting the presence or absenceof a nucleic acid mutation through the method of enzymatic nucleic acidamplification or ligation; wherein detection of a single nucleic acidmutation has >10% better signal discrimination compared to real-timePCR.

9. A sequence detection system able to accurately detect the absoluteconcentration of a target nucleic acid using the method of (A) producingmicrodroplets within an immiscible fluid in a capillary tube, wherein aplurality of microdroplets containing a single nucleic acid templatefrom the plurality of nucleic acid targets is formed; (B) amplifying thesingle nucleic acid template in the microdroplets by heating and coolingsuch that a plurality of single nucleic acid templates within themicrodroplets are amplified; and (C) detecting the presence or absenceof a target nucleic acid through the method of fluorescently detecting asignal generated by an enzymatic nucleic acid amplification or ligationreaction within the intact droplet; wherein detection of the absoluteconcentration of the target nucleic acid has >10% better quantitativeresolution compared to real-time PCR or quantitative PCR, and/or anadjustable quantitative resolution based on the total number of dropletsand target nucleic acid molecules processed.

10. A sequence detection system able to accurately detect theconcentration of a target nucleic acid using the method of (A) producingmicrodroplets within an immiscible fluid in a capillary tube, wherein aplurality of microdroplets containing a single nucleic acid templatefrom the plurality of nucleic acid targets is formed; (B) amplifying thesingle nucleic acid template in the microdroplets by heating and coolingsuch that a plurality of single nucleic acid templates within themicrodroplets are amplified; and (C) detecting the presence or absenceof a target nucleic acid through the method of fluorescently detecting asignal generated by an enzymatic nucleic acid amplification or ligationreaction within the intact droplet; wherein detection of small changes(<40%) in the absolute concentration of a target nucleic acid within asample or between samples.

11. A sequence detection system able to detect a gene copy numbervariation using the method of (A) producing microdroplets within animmiscible fluid, wherein a plurality of microdroplets containing asingle nucleic acid template from the plurality of nucleic acid targetsis formed; (B) amplifying the single nucleic acid template in themicrodroplets by heating and cooling such that a plurality of singlenucleic acid templates within the microdroplets are amplified; and (C)detecting the number of gene insertions or deletions in a genome throughthe method of counting the number of PCR amplicons of the target generelative to the number of PCR amplicons of a reference gene having aknown number of gene copies per genome; wherein detection of a targetgene copy number per genome has better signal discrimination compared torelative quantification (delta cycle threshold or delta delta cyclethreshold) by real time PCR in its ability to discriminate single copydifferences where the number or copies of the target gene is greaterthan 2 but less than 20.

12. A sequence detection system able to detect a low abundant singlenucleotide mutation using the method of (A) producing microdropletswithin an immiscible fluid, wherein a plurality of microdropletscontaining a single nucleic acid template from the plurality of nucleicacid targets is formed; (B) producing microdroplets within an immisciblefluid, wherein a plurality of microdroplets containing a single nucleicacid template from the plurality of nucleic acid targets is formedwherein in partitioning the sample reduces the ratio of target nucleicacid to competing background nucleic acids; (C) amplifying the singlenucleic acid template in the microdroplets by heating and cooling suchthat a plurality of single nucleic acid templates within themicrodroplets are amplified; and (D) detecting a single nucleotidemutation in a genetic sequence; wherein detection of a single nucleotidemutation has at least ten times better signal discrimination compared byreal time PCR in its ability to detect a mutant genome possessing asingle point mutation where the relative concentration of mutant geneticsequence is less than or equal to 0.1° A of the wild type genome.

(xi). Miscellaneous 3

1. A method of performing asynchronous sequential high-throughput PCR,comprising (A) providing one or more biological samples; (B) dividingeach of the one or more samples into one or more droplets using one ormore droplet generators; (C) isolating and storing the one or moredroplets from each of the one or more samples, thereby forming a dropletpacket from each of the samples; and (D) sequentially selecting at leasta portion of each of the packets and causing the portion to a flowthrough a thermal cycling device.

2. The method of paragraph 1, wherein the method further includes atleast one of (A) random access, (B) result-driven, on-demandtriage/diagnostics, (C) asynchronous loading, (D) stat mode, (E) aflexible number of samples, (F) a flexible number of reagents, and (G)digital PCR.

3. An apparatus, comprising (A) an injection molded portion comprisingat least a channel for transporting a biological sample and a secondchannel for receiving a droplet carrier fluid, partitioning the sampleinto one or more sample droplets, and directing the droplets to anoutlet, and (B) an instrument portion comprising an inlet for receivingthe droplets from the outlet a thermal cycler, and a detector; whereintogether the injection molded and the instrument portions perform one ormore nucleic acid assays.

4. The apparatus of paragraph 3, further comprising at least one of adroplet generator, a bead blender, a low-cost disposable, and areservoir or holding coil at the outlet.

III. SAMPLE PREPARATION/CARTRIDGE

This Section describes exemplary systems for sample preparation,including cartridges for sample lysis and droplet generation.

It may be desirable to separate an enzymatic amplification system suchas a PCR-based DNA amplification system into disposable andnondisposable components, for example, by creating a disposablecartridge or other disposable vessel that would prepare and presentsamples to a nondisposable PCR instrument or other reader. Such aseparation could facilitate rapid and low-cost DNA testing and analysis.The disposable cartridge may be designed as a single-use cartridge, toavoid the possibility of cross contamination between samples. Althoughthe terms “cartridge” or “disposable cartridge” will be used toreference the disposable portion of the DNA amplification system, thedisposable portion generally may take various forms, and need not berectangular or symmetric in any particular manner or dimension.

A suitable disposable cartridge will be configured to receive a sampleand to prepare (or at least partially prepare) the sample foramplification and analysis, prior to PCR thermocycling andamplification. The cartridge may include an interface configured to passthe prepared sample to a non-disposable portion of the system, whichgenerally will be referred to as an “instrument,” for subsequent PCRamplification and analysis steps. In some cases, the interface betweenthe cartridge and the instrument also may be configured to transfervarious fluids, such as oil and/or aqueous fluid, from the instrument tothe cartridge, to “prime” or partially prime the cartridge for samplepreparation. In other cases, the cartridge may be partially or entirelypre-primed with fluids, so that fluid transfer from the instrument isnot necessary.

A disposable cartridge according to the present disclosure may beconfigured to generate droplets or packets of droplets, each containinga mixture of sample and reagent, which then may be transported from thedisposable cartridge to the related instrument for rapid serialinjection into a continuous flow thermal cycler. The cartridge or otherdisposable vessel then may be removed from the system and discarded. Thecartridge may be configured to perform sample preparation stepsrelatively quickly, as measured by sample throughput from the cartridgeto the PCR instrument. For example, a cartridge according to the presentdisclosure may be configured to perform sample preparation in a time ofless than 5 minutes per sample, to achieve throughput of at least 10samples per hour. The cartridge also may be constructed from andfunction in conjunction with non-hazardous materials, to minimizeenvironmental impact.

FIG. 41 is a flowchart depicting the steps of a DNA amplificationmethod, generally indicated at 1600, that may be performed within or inconjunction with a disposable cartridge of a DNA amplification systemaccording to the present disclosure. The major functions that thedisposable cartridge is configured to perform are purification, lysis,reagent mixing, and sample isolation into droplets. However, moregenerally, any subset or combination of the steps depicted in FIG. 41may be performed within the cartridge. Alternatively, one or more of thedepicted steps, such as sample collection and extraction, may beperformed prior to transferring target-containing material into thecartridge, while other steps are performed within the cartridge.Similarly, one or more of the depicted steps, such as dropletgeneration, may be performed after transferring target-containingmaterial out of the cartridge. Furthermore, the steps depicted in FIG.41 may be performed in various different orders, only some of which willbe described below.

At step 1602 of method 1600, a sample is collected for subsequentanalysis. This is typically done by a medical practitioner, a lawenforcement agent, a scientist, or some other person with reason tocollect a sample for nucleic acid analysis. The sample may, for example,be collected using a sample collector, such as a swab, a sample card, aspecimen drawing needle, a pipette, a syringe, and/or by any othersuitable method. Furthermore, pre-collected samples may be stored inwells such as a single well or an array of wells in a plate, may bedried and/or frozen, may be put into an aerosol form, or may take theform of a culture or tissue sample prepared on a slide. Suchpre-collected samples then may be obtained and prepared fordroplet-based processing in a disposable cartridge. The collected sampletypically will include one or more cells, bacteria, viruses, or othermaterial potentially or actually containing a target sequence ofnucleotides suitable for PCR amplification.

At step 1604, the collected sample is extracted from the samplecollector. This may be accomplished, for example, by transferring thesample from the sample collector using a pipette, a syringe, or thelike, or by soaking and/or rinsing a sample collector in one or moresuitable solutions, such as a digestive buffer solution, a lysis buffersolution, or an appropriate binder-containing solution, among others.Extraction may occur within a chamber of the disposable portion of thePCR system, in which case the sample will be transferred to thecartridge, as indicated at step 1606 of method 1600, prior toextraction. Alternatively, extraction may occur outside of thecartridge, and the resulting sample or sample-containing solution thenmay be transferred to the cartridge. In either case, the cartridge maybe configured to perform various additional sample preparation steps, asdescribed below.

At steps 1608 and 1610, the extracted sample, which is now disposed in asample chamber within the cartridge, is purified and lysed. These stepsmay be performed at different times, simultaneously, or approximatelysimultaneously. Furthermore, purification may be performed either beforeor after lysing, and in some instances two or more separate purificationsteps may be performed, one before lysing and one after lysing.Purification generally includes some form of filtering to removeunwanted components from the sample while leaving the desired targetcomponents relatively unaffected, and lysing generally includesdisruption of the sample constituents (e.g., by breaking the cellularmembranes) to expose target DNA for amplification, typically involvingsome form of physical blending or stirring of the sample-containingmixture. For example, lysing may proceed through bulk mixing such asagitation, magnetic stirring, and/or aspiration, or through microfluidicmixing of various types such as forcing the sample through a tortuouspath, electromagnetic bombardment, sonication, and/or convection. Thefluid containing the contents of the lysed sample may be referred to asa lysate.

Depending on whether a particular purification step is performed beforeor after lysing, the method of purification may vary. For example,purification prior to lysing may be configured to capture relativelylarge target-containing material, such as bacteria or other cells.Purification at this stage may, for example, include filtering thesample-containing solution through an aperture-based filter with acharacteristic aperture size smaller than the characteristic size of thetarget-containing cells, to retain the cells or other target materialwithin the sample chamber while removing other, smaller waste material.On the other hand, purification after lysing may be configured tocapture relatively small target material, such as DNA or partial nucleicacid sequences. Accordingly, post-lysing purification may includefiltration through a smaller filter, and/or affinity capture of DNA orother target material, to retain target material within the sample whileremoving other, larger waste material. In some cases, such as whenpurification steps are performed both before and after lysing, two ormore different types of filters, including aperture-based filters and/oraffinity-based filters, may be used.

At step 1612, the partially processed sample (i.e., the lysate) isconcentrated. This step is generally accomplished by separating excessfluid in the lysate from the target DNA or DNA-containing material, forexample, by filtering, ethanol precipitation, butanol extraction, oraffinity capture, among others. In any case, the result of theconcentration step is a greater density of target material per unitvolume of fluid. Concentration of the sample at this stage may result ina detectable amplified target after relatively fewer PCR amplificationcycles than would be necessary without concentration.

At step 1614, a PCR reagent mixture including appropriate enzymes andDNA primers is mixed with the sample. These reagent constituents areselected to facilitate DNA amplification of a particular target inconjunction with cyclical temperature changes (i.e., thermocycling). Thereagent mixture may be combined with the sample in fluid form, or it maybe lyophilized (freeze-dried) and converted into a powder, a pellet, orany other convenient form. To form a lyophilized reagent, suitablestabilizing and/or sedimenting agents may be combined with the PCRenzymes and DNA primers.

Two or more reagents may be mixed with the sample at step 1614, to formeither a single sample/reagent mixture containing multiple reagents, ormultiple mixtures each containing a single reagent. A single mixturecontaining multiple reagents may, for example, allow screening formultiple targets simultaneously, whereas multiple mixtures eachcontaining a single reagent may be configured for PCR amplification ofseveral different DNA targets, or (when two or more of the mixturescontain the same reagent) to provide experimental control, for instance,by allowing multiple PCR amplification and/or detection techniques to beapplied to the same sample/reagent mixture. When multiple sample/reagentmixtures are used, the different mixtures may be separately preparedand/or separately tracked through the system.

At step 1616, droplets containing the sample and the reagent aregenerated, typically in aqueous form within an oil-based emulsion. Thegenerated droplets may contain a mixture of sample and reagent, eitheractivated or not activated (i.e., either requiring or not requiring anadditional activation step before PCR amplification begins), or thedroplets each may contain sample and reagent that are separated fromeach other, for example, by a thin membrane, such as an oil membrane.When more than one sample/reagent mixture is present, dropletscontaining each of the various mixtures may be separately produced andtracked. Common modes of droplet generation include flow focusing,jetting, and shearing. Using these techniques, stable droplets may becreated at throughputs of 10-1000 Hz with tunable volumes ranging from15 picoliters (pL) to 5 nanoliters (nL). Various techniques forgenerating droplets are known.

At step 1618, the droplets produced in step 1616 are transferred fromthe disposable cartridge to a non-disposable instrument portion of thesystem. As noted above, the droplets may be contained within anemulsion, such as an oil-based emulsion, in which case transferring thedroplets will include transferring portions or the entirety of theemulsion. When more than one sample/reagent mixture has been created,the droplets containing each type of mixture may be separatelytransferred in a continuous or semi-continuous manner, so that eachseparate droplet type can be separately processed by the instrumentportion of the system. Continuous or semi-continuous droplet transfermay allow relatively rapid screening for multiple target DNA segments.Alternatively, or in addition, droplets containing varioussample/reagent mixtures may be “tagged” in some manner, such as with abar code or some other detectable component, in which case differenttypes of droplets may in some instances be transferred to thenon-disposable portion of the system together and then tracked ordetected individually.

After transfer from the disposable, sample-preparation cartridge portionof the PCR system to the non-disposable instrument portion,thermocycling and analysis will occur. The following examples describespecific exemplary methods and apparatus for receiving a sample in adisposable vessel, such as a cartridge, preparing the sample for PCRamplification, and passing the prepared sample to a reusable instrumentportion of a PCR amplification system. Additional pertinent disclosuremay be found in the U.S. provisional patent applications listed aboveunder Cross-References and incorporated herein by reference,particularly Ser. No. 61/277,249, filed Sep. 21, 2009.

A. Example 1: Disposable Sample Cartridge 1

This example depicts a disposable sample preparation cartridge andsuitable fluidic connections between various components of thecartridge; see FIG. 42.

FIG. 42 is a schematic view of the cartridge, generally indicated at1700, and suitable fluidic connections between various components of thecartridge. Cartridge 1700 is configured to receive and prepare atarget-containing sample for PCR thermocycling and amplification.Preparation of the sample may include some or all of the following steps(not necessarily in this order): purification, lysing, concentration,combination with one or more reagents, and/or generation of dropletssuitable for PCR. Droplets containing sample and reagent may betransferred from the cartridge to an instrument, generally indicated at1700′, which is configured to heat the droplets cyclically to facilitatePCR amplification. Dashed line L in FIG. 42 represents the interfacebetween disposable cartridge 1700 and instrument 1700′. This interfacemay include suitable fluidic connectors, receptors, and the like, toprovide a reliable fluidic connection between the cartridge andinstrument without significant leakage or contamination.

A sample chamber 1702 of cartridge 1700 is configured to receive asample. The sample entering chamber 1702 will contain, or at leastpotentially contain, a target for PCR amplification, such as one or morebacteria, viruses, DNA molecules, and/or other material that containsnucleic acid sequences. For example, the sample may be loaded in theform of eluant that was prepared from a sample collection swab. In somecases, the sample transferred to chamber 1702 may already have beenprepared to some extent, for example, by washing, concentrating, and/orlysing, and in other cases the sample may be substantially unprepared or“raw” when it reaches chamber 1702. In any case, sample chamber 1702 maybe configured to receive and prepare the sample as described below.

A waste chamber 1704 is fluidically connected to sample chamber 1702,and cartridge 1700 is configured to transfer fluid out of sample chamber1702, through a filter 1706, and into the waste chamber. Filter 1706 isconfigured to allow waste products to pass through itself and into thewaste chamber, while retaining the PCR target material within the samplechamber. For example, filter 1706 may be a membrane or other similaraperture-type filter with a known characteristic size cutoff.Alternatively, or in addition, the filter may be configured to retainthe PCR target within the sample chamber through a suitable form ofaffinity capture, such as by coating a portion of the sample chamberwith an appropriate binding compound. The filter may be used to captureand pre-concentrate the target before the sample is washed, and/or itmay be used to retain, additionally concentrate, and/or purify thesample after the sample is washed.

A reservoir chamber 1708 is fluidically connected to sample chamber1702, and is configured to transfer to the sample chamber areconstitution fluid, a wash solution, and/or any other fluid suitablefor combination with the filtered sample. For example, the fluidtransferred from the reservoir chamber may be water, or a buffersolution, such as TE buffer (i.e., a combination oftris(hydroxymethyl)aminomethane, hydrochloric acid, and EDTA), which mayremove matrix components that could inhibit downstream PCRamplification. Fluid transferred from the reservoir chamber generallymay include any agent configured to separate the target from undesirablecomponents that may have been originally attached to the sample or thatmay have been used to capture the target when filter 1706 operatesthrough affinity capture.

Sample chamber 1702 also may be configured to lyse the sample. Lysingwill typically, but not necessarily, be performed after the target hasbeen washed and/or reconstituted with fluid transferred from reservoirchamber 1708. Lysing may be performed within the sample chamber throughmechanical agitation, such as blending, vibrating, shaking, and/orstirring the sample within the chamber, to release nucleic acids fromthe sample. In some cases, agitation elements, such as discs, rods,and/or small beads may be present in the sample chamber to facilitatelysing. The sample and/or the agitation elements may be agitated by anysuitable method, such as manually, through the application of soundwaves (i.e., sonication), and/or using magnetic or electromagneticforces.

Sample chamber 1702 also may be configured to concentrate thetarget-containing fluid sample. This can be accomplished prior towashing, by transferring some of the original sample-containing fluidfrom the sample chamber, through the filter, and into the waste chamber.Alternatively, or in addition, concentration can be accomplished bytransferring some of the sample-containing fluid into the waste chamberafter the sample is washed, while completely or substantially retainingthe target nucleic acids within the sample chamber. Concentrating thefluid sample in this manner results in a greater number of targetnucleic acids per unit volume of fluid, which can lead to more efficientand faster PCR amplification in subsequent processing steps.

Cartridge 1700 includes one or more reagent chambers. Two reagentchambers 1710 a, 1710 b are depicted in FIG. 42, but more generally anydesired number of reagent chambers, such as five or more, may beutilized. Each reagent chamber contains reagents, such as primers,polymerase, and appropriate enzymes, configured to react with aparticular target nucleic acid sequence and to undergo PCR amplificationif the target is present in the sample. Typically, the reagents will bepre-loaded into each reagent chamber during the cartridge manufacture,although in some embodiments the reagents may be loaded by a user ortransferred from a related PCR instrument.

The reagents may be stored in or introduced into the reagent chambers inany suitable manner. For example, the reagents may take the form oflyophilized pellets 1711 a, 1711 b depicted in FIG. 42, or a coating(not shown) applied to a portion of the interior wall of each reagentchamber. Alternatively, a reagent coating may be applied to a stirelement disposed within the reagent chamber, and/or to a plunger used tovary transfer fluid into and out of the reagent chamber. The reagentchambers of FIG. 42 are fluidically connected in parallel with thesample chamber, so that each reagent chamber can separately receive aportion of the filtered, lysed sample-containing solution, withoutcross-contamination. One or more stir elements (not shown) may beincluded in each reagent chamber to facilitate mixing the sample withthe pre-loaded reagents. When stir elements are included in the reagentchambers, they may operate manually, through sonication, or usingmagnetic or electromagnetic forces, in a manner similar to the operationof the agitation elements used for lysing in the sample chamber.

Reagent chambers 1710 a and 1710 b are each fluidically connected to adroplet generator, generally indicated at 1712. Droplet generator 1712is configured to generate discrete micro-volume droplets, eachcontaining all of the ingredients for subsequent nucleic acidamplification via PCR. In general, droplet generator 1712 is configuredto generate one or more water-in-oil emulsions, although other types ofemulsions, such as oil-in-water, water-in-oil-in-water, and so forth arealso possible.

Parallel fluid connections lead to droplet generator 1712 from reagentchambers 1710 a and 1710 b. A common oil reservoir 1714 is configured totransfer oil along the fluid paths indicated, so that oil arrives ateach of intersection points 1716 a and 1716 b from two separatedirections. At the intersection points, sample-containing solutionarrives from the respective reagent chambers and combines with the oilfrom the oil reservoir to form water-in-oil droplets. The generateddroplets are then transferred across interface L and into instrument1700′. Each sample/reagent mixture may be transferred either serially orin parallel to droplet generator 1712. Other droplet generatorconfigurations may be suitable, as described below.

After droplets have been generated, system 1700 is configured tofacilitate transfer of the droplets through interface L to instrument1700′. This transfer may be accomplished through the use of suitablefluidic tubing, capillaries, pumps, valves, and/or the like, which maybe configured to transfer droplets to the instrument either as parallelstreams or in separate (serial) batches, each of which contains dropletsthat include a specific reagent. The droplets then may be transferredthrough a multi-port valve and introduced into a thermocycler for PCRamplification.

B. Example 2: Disposable Sample Cartridge 2

This example describes an exemplary disposable cartridge that issuitable for performing some or all of the sample preparation stepsdescribed above; see FIGS. 43-45.

FIG. 43 is an isometric view of an interior portion of the exemplarycartridge, generally indicated at 1720. The cartridge is configured tointerface with an instrument (not shown), so that prepared samples canbe transferred to the instrument, generally in the form of awater-in-oil emulsion, for PCR amplification and analysis. In additionto the interior portion depicted in FIG. 43, cartridge 1720 also mayinclude a suitable exterior housing (not shown) disposed around some orthe entirety of the interior portion. The exterior housing may beconfigured to protect the interior portion and may be shaped tofacilitate storage and/or transportation of multiple cartridges.

Cartridge 1720 includes an upper section 1722 and a lower section 1724,which are configured to fit together to form the interior portion of thecartridge. For clarity, the upper and lower sections are separated by aslight gap in the drawings. These sections may be manufactured by anysuitable method, such as by injection molding a thermoplastic material.The upper and lower sections may be bonded together in any suitablemanner, for example, with connecting pins (or similar connectors), withan adhesive, and/or by thermal curing, to maintain the structuralintegrity of the assembled cartridge.

FIGS. 44 and 45 are side elevation and top views, respectively, of theinterior portion of cartridge 1720. These drawings, together with FIG.43, show that the cartridge includes a number of discrete chambers.These chambers are fluidically connected by a fluid path, which isgenerally indicated at 1726 in FIG. 45. Fluid path 1726 may result fromjoining complementary grooves formed within each of sections 1722 and1724, so that a closed fluid path results when the sections are joinedtogether. The grooves of each section may, for example, have anapproximately hemispherical profile, so that the grooves form asubstantially cylindrical fluid path when the upper and lower sectionsof the cartridge are assembled. In other embodiments, the grooves mayhave other shapes, such as rectangular, and the allocation of the totalcross section between the upper and lower sections may vary.

A sample chamber 1728 of cartridge 1720 is configured to receive asample that contains (or potentially contains) a target nucleic acidsequence. The sample may be transferred into the sample chamber as afluid, or it may be placed in the chamber attached to a swab or someother suitable sample collection medium. The sample chamber can beconstructed to have any desired shape, such as the cylindrical shapedepicted in FIGS. 43 and 44, and any desired volume, such as a volume inthe range of 200 microliters (4) to 2 milliliters (mL). The volume ofthe sample chamber may depend in part on the number of separate nucleicacid targets for which the cartridge is configured to test, as describedbelow.

Sample chamber 1728 may include a filter 1730. The filter will typicallybe disposed near or below the bottom surface of the sample chamber.Filter 1730 may be a size-exclusion filter configured to prevent passageof material larger than a particular preselected size. For example, toprevent passage of bacteria having a characteristic size of 600nanometers (nm), the filter may be a membrane with a characteristiccutoff size of 200-400 nm. To prevent passage of other material, thefilter may be chosen to have a different characteristic cutoff size,which is selected based on the material to be filtered. Membranefiltration based on size fractionation is a simple, yet effective methodof capturing target cells. Once captured, the cells can be washed toremove potential PCR inhibitors that are soluble or below the sizecutoff of the membrane.

Alternatively, filter 1730 may operate through affinity capture (i.e.,by attracting and/or chemically binding one or more target molecules),or by solid phase extraction, such as chemical precipitation. However,membrane filtration may have certain advantages over solid phaseextraction, including a reduced number of processing steps, no hazardousreagents, fast processing times, and the potential for simultaneousconcentration and purification of the target organisms, as describedbelow.

The sample chamber also may include one or more lysing elements, such asa stirring disc 1732 and/or lysis beads 1734; see FIGS. 43-44. Theseelements are generally configured to facilitate lysis of a fluid in thesample chamber, through agitation of the sample to release nucleic acidsby breaking down surrounding material (such as cellular material). Thelysing disc 1732 or other similar stirring element will typically bedisposed toward the bottom of, but within, the sample chamber. Lysisbeads 1734, which can take the form of beads of any desired material anddiameter, such as glass beads with diameters in the range of 70-700 μm,are configured to further facilitate lysis by colliding with anddisrupting material within the agitated fluid of the sample chamber.

Agitation of stirring disc 1732, which also can take the form of a rodor any other suitable shape, may be provided by magnetic orelectromagnetic forces. For example, the stirring disc may besufficiently magnetic to respond to a changing magnetic field applied tothe sample chamber. Thus, variations in the applied magnetic field cancause the stirring disc to spin and/or tumble, resulting in agitation ofthe fluid within the sample chamber. A variable magnetic field may beprovided, for example, by a single low-cost driver located on therelated PCR instrument. The driver may be configured to drive the lysingelements within one, several, and/or a multitude of sample chamberssimultaneously. Because the lysing elements are contained within thesample chamber and because the magnetic driver may be configured to actacross a plurality of sample chambers, lysing within cartridge 1720 doesnot require a special interface between the disposable cartridge and therelated instrument. This configuration provides a high degree ofamenability to integration and automation within a low-cost single-usecartridge.

Sample chamber 1728 is configured to receive one or more fluids, such asa wash and/or a reconstitution solution, from a reservoir chamber 1736.When the sample transferred to the sample chamber is attached to amedium, such as a swab, fluid from the reservoir chamber may be used toreconstitute the sample into fluidic form. Fluid from the reservoirchamber also may be used to purify a sample, such as bacteria, bywashing the sample with a buffer solution. The fluid in reservoirchamber 1736 may be provided with the cartridge, supplied by a user,and/or transferred to the cartridge from an instrument to which thecartridge attaches. In any case, fluid may be transferred from reservoirchamber 1736 to sample chamber 1728 along fluid path 1726, whichconnects the two chambers. This connection can be seen, for example, inFIG. 45, which is a top view of cartridge 1700. Fluid transferred fromthe reservoir chamber to the sample chamber passes through filter 1730,so that the fluid is filtered before entering the sample chamber.

Cartridge 1720 also includes a waste chamber 1738. The waste chamber isconfigured to receive waste material, such as nucleic acid fragments andother waste material either introduced to the sample chamber with thesample or fragmented during lysing, from the sample chamber. Wastechamber 1738 is fluidically connected to sample chamber 1728 throughfluid path 1726, which passes through filter 1730. Accordingly, fluidand fragmentary waste products may be transferred from the samplechamber to the waste chamber, while target material having acharacteristic size (or chemical affinity) suitable for capture by thefilter will be retained within the sample chamber.

For example, sample-containing solution may be purified prior to lysingby filtering the fluid through filter 1730 and into waste chamber 1738.The fluid in the sample chamber then may be replenished from reservoirchamber 1736, as described previously. Similarly, sample-containingsolution may be purified and/or concentrated after lysing, again byfiltering the fluid through filter 1730 and into waste chamber 1738. Thesteps of purification, concentration, and fluid replenishment may berepeated any desired number of times by transferring fluid from thesample chamber to the waste chamber and from the reservoir chamber tothe sample chamber.

FIGS. 43-45 depict five separate reagent chambers 1740 a, 1740 b, 1740c, 1740 d and 1740 e within cartridge 1720. In general, any desirednumber of reagent chambers, from one, two, three, four, five, six,seven, eight, nine, ten, or more, up to an arbitrarily large number, maybe provided (both in this embodiment and other disposable cartridgesshown herein). Each reagent chamber is configured to receivesample-containing fluid from the sample chamber, and to allow thecombination of the sample-containing fluid with a particular reagentmixture. Sample-containing fluid can be transferred from the samplechamber to the reagent chambers along fluidic path 1726, which connectsthe sample chamber to each of the reagent chambers in parallel, as canbe seen in FIG. 45.

Each reagent mixture may include, for example, primers, polymerase,and/or enzymes suitable for PCR amplification of a particular nucleicacid sequence. The reagent mixtures in two or more of reagent chambers1740 may be the same or substantially similar (for example, to allow forexperimental control), or each reagent mixture may be substantiallydifferent, to search for multiple different target nucleic acidsequences.

The reagent mixtures of cartridge 1720 are depicted as lyophilizedpellets 1742 a, 1742 b, 1742 c, 1742 d, and 1742 e disposed at thebottom of the associated reagent chambers; see FIG. 45. However, ingeneral the reagent mixtures can be provided in any suitable form, suchas within a fluid, as a lyophilized powder (either loose or shaped intoa form other than a pellet), or as a coating applied to the interiorsurface of each reagent chamber, among others. Furthermore, the reagentmixtures may be supplied with the cartridge, supplied by a user, ortransferred to the cartridge from a PCR instrument to which thecartridge is connected.

Cartridge 1720 also includes an oil chamber 1744, which is fluidicallyconnected to each of reagent chambers 1740 a, 1740 b, 1740 c, 1740 d,and 1740 e. Oil chamber 1744 is configured to supply the oil needed toproduce a water-in-oil emulsion containing droplets of sample andreagent fluid. More specifically, oil can pass from chamber 1744 to aplurality of droplet generation regions 1745 a, 1745 b, 1745 c, 1745 d,and 1745 e, each corresponding to and fluidically connected with one ofthe reagent chambers. Each droplet generator is configured to generatedroplets of a particular sample/reagent mixture suspended in an oilbackground.

Specifically, as depicted in FIG. 45, oil in cartridge 1720 passes fromoil chamber 1744 down a plurality of fluid pathways. These include apair of oil pathways corresponding to each droplet generator andconfigured to intersect with a fluid pathway from one of the reagentchambers, to create water-in-oil droplets. The generated droplets thenmay pass through interface components, such as a plurality of capillaryconnectors 1746 a, 1746 b, 1746 c, 1746 d, and 1746 e. The capillaryconnectors are configured to transfer fluid to a plurality ofcorresponding capillaries 1748 a, 1748 b, 1748 c, 1748 d, and 1748 e,which are configured to interface with instrument 1700′ (see, e.g., FIG.42).

C. Example 3: Exemplary Hydraulic Mechanisms

This example describes aspects of two exemplary hydraulic mechanismssuitable for controlling fluid motion between the various chambers of adisposable cartridge; see FIGS. 46 and 47.

FIG. 46 schematically illustrates aspects of a two-chamber hydraulicmechanism, generally indicated at 1760, that is suitable for controllingfluid motion between the various chambers of a disposable cartridge,such as cartridges 1700 or 1720 described above. Each side of FIG. 46depicts two fluid chambers 1762 and 1764. Each chamber is equipped witha plunger 1766, and a fluid 1768 is partially disposed within eachchamber. In the left-hand portion of FIG. 46, the majority of the fluidis disposed in chamber 1764, and in the right-hand portion of FIG. 46,the majority of the fluid is disposed in chamber 1762. A connectingfluid pathway 1770 is provided between chambers 1762 and 1764, whichallows fluid 1768 to pass between the chambers.

Fluid motion between chambers will occur when unequal forces are appliedto the two plungers 1766, causing one of the plungers to move down whilethe other moves up. Such forces will typically be applied by a forceactuator, such as a piston or a push rod, which will be contained withinor otherwise integrated with an instrument configured to receive adisposable sample preparation cartridge. In this manner, fluid can betransferred between any of the previously described chambers of adisposable cartridge in a controlled manner.

More specifically, motions of plungers 1766 may be controlled directlyby a user and/or by an instrument configured to receive and interactwith the cartridge containing the plungers. For example, a user mightmanually load a sample or a sample-containing fluid into one of chambers1762 or 1764 (which would therefore be considered a sample chamber), andthen insert a plunger 1766 into the chamber, sealing the sample orsample-containing fluid within the chamber. Fluid then may betransferred hydraulically into and out of the sample chamber bydepressing the appropriate plunger either manually or automatically.

Automatic plunger motions may be controlled by a processor programmed totransfer fluids between chambers of the system in a predeterminedmanner. For instance, if hydraulic mechanism 1760 is incorporated intocartridge 1700, then instrument 1700′ may include force actuatingstructures complementary to the plungers of the hydraulic mechanism,such as pistons, push rods or the like. These force actuators may beconfigured to depress the associated plungers at particular times, in aparticular order, or in response to signals sent to the instrument by auser.

FIG. 47 schematically depicts a three-chamber hydraulic mechanism,generally indicated at 1780, which is similar to two-chamber mechanism1760 of FIG. 46. Fluid chambers 1782, 1784, and 1786 each include aplunger 1787. A fluid 1788 is partially disposed within each chamber,and the chambers are fluidically connected by a fluid pathway 1790.Accordingly, fluid will be transferred from one chamber to one or bothof the other chambers when plungers 1787 are moved appropriately. Forexample, fluid from chamber 1786 can be transferred to chambers 1782 and1784 by depressing the plunger of chamber 1786 and simultaneouslyraising the plungers of chambers 1782 and 1784.

If the chambers all have the same size and geometry, then to transfer anequal amount of fluid from chamber 1786 to chambers 1782 and 1784, eachof the plungers of chambers 1782 and 1784 would be raised at half therate with which the plunger of chamber 1786 is depressed. Alternatively,the chambers may have different sizes and/or shapes, in which case theplunger motions would be suitably modified to achieve equal fluidtransfer from one chamber to the other chambers. Furthermore, fluid fromone chamber can be divided among two or more other chambers according toany desired ratio of volumes, by controlling the motions of the variousplungers.

Plungers according to the present disclosure may include a lockingmechanism. The locking mechanism of a particular plunger may beconfigured to lock the plunger into a particular position, to avoidundesirable transfer of fluid to or from a particular chamber. Forexample, a plunger associated with a waste chamber may include a lockingmechanism configured to lock the plunger in place when the plungerreaches an upper (retracted) position, corresponding to a maximum volumeof fluid within the waste chamber. This can prevent waste fluid fromunintentionally being transferred back into another chamber, such as asample chamber or a reservoir chamber, after waste has been removed froma sample.

A suitable plunger locking mechanism can take various forms, each havingthe common property that the mechanism prevents particular unwantedplunger motions. For example, a suitable locking may include a mechanismintegrated with the plunger itself, such as a spring-biased tab or thelike (not shown) that snaps into place when the plunger reaches acertain position, preventing subsequent downward plunger motions.Alternatively, the locking mechanism may be associated with theinstrument configured to receive the disposable cartridge, in which casethe locking mechanism may include programming a controller to avoidcausing downward motions of a particular plunger under certaincircumstances.

Plungers according to the present disclosure also may be configured tolimit or eliminate leaks. For example, as depicted in FIG. 47, plungers1787 may include both a lower seal 1790 and an upper seal 1792, attachedto a common shaft 1794 and separated by a desired distance. Seals 1790and 1792 typically will take the form of o-rings or similar structuresconfigured to fit in a substantially fluid-tight manner within the innercircumference of the associated chamber. Thus, as FIG. 47 depicts (seechamber 1786), any residual fluid 1788 that passes the lower seal as aplunger is depressed will still be trapped within the associated chamberby the upper seal.

D. Example 4: Exemplary Droplet Generators

This example describes various exemplary droplet generationconfigurations that may be suitable for generating water-in-oil dropletscontaining a mixture of sample and reagent; see FIGS. 48A-48F. Thegenerated droplets then may be transported to a thermocycling instrumentfor PCR amplification. Each depicted configuration is compatible withcontinuous production of oil phase emulsions and with bothpressure-controlled and positive displacement pumping. A dropletgenerator or droplet generation configuration according to the presentdisclosure may be connected to a pressure/pump source located on acomplementary PCR instrument, or may include any pumps and/or pressuresources needed to facilitate droplet generation.

Each depicted droplet configuration in FIGS. 48A-48F may be capable ofhigh-throughput droplet generation (˜1,000 droplets per second) in adisposable device, such as a cartridge. Each configuration may beconstructed by injection molding two layers of material that fittogether to form fluid channels, such as cylindrical channels formed bycomplementary hemispherical grooves. The fluid channels of the dropletgeneration configurations depicted in FIGS. 48A-48F may have varyingchannel depths, such as 50, 100, 150, 200, or 250 μm, among others.

FIG. 48A depicts a 3-port cross droplet generation configuration 1800wherein oil from a first fluid well (or chamber) 1802 is transferredthrough two similar branches of a fluid channel section 1804. The oilfrom well 1802 intersects with aqueous fluid from a second fluid chamber1806, which is transferred along a fluid channel section 1808 to anintersection area generally indicated at 1810. The oil from well 1802arrives at intersection 1810 from two different and substantiallyopposite directions, whereas the aqueous solution arrives at theintersection along only a single path that is substantiallyperpendicular to both directions of travel of the arriving oil. Theresult is that at intersection 1810, aqueous droplets in an oilbackground (i.e., a water-in-oil emulsion) are produced and transferredalong a fluid channel section 1812 to a third chamber 1814, where theemulsion can be temporarily stored and/or transferred to a thermocyclinginstrument.

FIG. 48B depicts a configuration 1815 that is similar in most respectsto droplet generation configuration 1800 depicted in FIG. 48A.Specifically, in droplet generation configuration 1815, oil from a firstfluid chamber 1816 is transferred through two similar branches of afluid channel section 1818. Fluid channel sections 1818 intersect with afluid channel section 1822 that transfers aqueous fluid from a secondfluid chamber 1820, at an intersection area generally indicated at 1824.As in configuration 1800, the oil from chamber 1816 arrives atintersection 1810 from two different directions, but unlike inconfiguration 1800, the oil does not arrive from substantially opposite(antiparallel) directions. Rather, channel sections 1818 each intersectchannel section 1822 at a non-perpendicular angle, which is depicted asapproximately 60 degrees in FIG. 48B. In general, configuration 1815 mayinclude oil fluid channels that intersect an aqueous fluid channel atany desired angle or angles. Oil flowing through channel sections 1818and aqueous solution flowing through channel section 1822 combine toform a water-in-oil emulsion of aqueous droplets suspended in an oilbackground. As in the case of configuration 1800, the droplets then maybe transferred along a fluid channel section 1826 to a third fluidchamber 1828, for storage and/or transfer to a thermocycling instrument.

FIG. 48C depicts a four-port droplet generation configuration 1829 thatincludes two separate oil wells or chambers. A first oil chamber 1830 isconfigured to store oil and transfer the oil through a fluid channelsection 1832 toward a channel intersection point generally indicated at1842. A second oil chamber 1834 is similarly configured to store andtransfer oil toward the intersection point through a fluid channelsection 1836. An aqueous fluid chamber 1838 is configured to storeaqueous fluid, such as a sample/reagent mixture, and to transfer theaqueous fluid through fluid channel section 1840 toward intersectionpoint 1842. When the oil traveling through fluid channel sections 1832and 1836 intersects with the aqueous fluid traveling through fluidchannel section 1840, a water-in-oil emulsion of aqueous dropletssuspended in oil is generated. Although fluid channel 1840 is depictedas intersecting with each of fluid channels 1832 and 1836 at aperpendicular angle, in general the channels may intersect at anydesired angle, as described previously with respect to dropletgeneration configuration 1815 of FIG. 48B. The emulsion generated atintersection 1842 travels through outgoing fluid channel section 1844toward an emulsion chamber 1846, where the emulsion may be temporarilyheld for transfer to an instrument, such as a thermocycling instrument.

FIGS. 48D-48F schematically depict fluid channel intersection regions ofseveral other possible droplet generation configurations, in which thearrows within the depicted fluid channels indicate the direction offluid flow within each channel. Although fluid chambers for receivingand/or storing oil, water, and any generated emulsion are not depictedin FIGS. 48D-48F, these chambers or at least some source of oil andaqueous fluid would be present in a cartridge containing any of thedepicted configurations. The fluid channels and any associated chambersmay be formed by any suitable method, such as injection moldingcomplementary sections of thermoplastic as described previously.

FIG. 48D depicts a “single T” configuration 1850 in which oil travelingin an oil channel 1852 intersects with aqueous fluid traveling in anaqueous channel 1854 at fluid channel intersection 1856, to produce awater-in-oil emulsion that travels through outgoing fluid channel 1858.This configuration differs from those of FIGS. 48A-48C in that oilarrives at the oil/water intersection from only a single direction.Accordingly, droplets may be formed by a slightly different physicalmechanism than in configurations where oil arrives from two directions.For example, droplets formed in the single T configuration of FIG. 48Dmay be formed primarily by a shear mechanism rather than primarily by acompression mechanism. However, the physics of droplet formation is notcompletely understood and likely depends on many factors, including thechannel diameters, fluid velocities, and fluid viscosities.

FIG. 48E depicts a “double T” configuration 1860 in which oil travelingin an oil channel 1862 intersects with aqueous fluid traveling in afirst aqueous channel 1864 at a first intersection 1866, to produce awater-in-oil emulsion that travels through intermediate fluid channel1868. Channel 1868 intersects with a second aqueous channel 1870 at asecond intersection 1872, to generate additional water-in-oil dropletswithin the emulsion. All of the generated droplets then travel throughoutgoing fluid channel 1874. This configuration again differs from thoseof FIGS. 48A-48C in that oil arrives at the oil/water intersections fromonly a single direction. In addition, configuration 1860 differs fromsingle T configuration 1850 depicted in FIG. 48D due to the presence oftwo oil/water intersections. This may result in a greater density ofdroplets in the water-in-oil emulsion generated by configuration 1860than in the emulsion generation by configuration 1850, which includesonly one oil/water intersection.

FIG. 48F depicts a droplet generation configuration 1880 in which oiltraveling in an oil channel 1882 intersects with aqueous fluid travelingin first and second aqueous channels 1884 and 1886 at an intersection1888. In this configuration, the aqueous fluid arrives at theintersection from two opposite directions, both of which aresubstantially perpendicular to the direction of travel of the oil inchannel 1882. More generally, the aqueous fluid can intersect with theoil at any desired angles. Depending on at least the sizes of thevarious channels, the flow rates of the oil and the aqueous fluid, andthe angle of intersection of the aqueous fluid channels with the oilchannel, a configuration of this type may be suitable for producingeither an oil-in-water emulsion or a water-in-oil emulsion. In eithercase, the emulsion will travel away from intersection 1888 throughoutgoing fluid channel 1890.

E. Example 5: Disposable Sample Cartridge 3

This example describes aspects of three alternative disposable samplepreparation cartridges; see FIGS. 49-51.

FIG. 49 is a schematic diagram depicting another disposable samplepreparation cartridge, generally indicated at 1900, and suitable fluidicconnections between various components of the cartridge. Cartridge 1900is configured to receive and prepare a target-containing sample for PCRthermocycling and amplification, and is substantially similar tocartridge 1700 depicted in FIG. 42 in many respects. Accordingly,cartridge 1900 includes a sample chamber 1902, a waste chamber 1904, afilter 1906, a reservoir chamber 1908, and reagent chambers 1910 a, 1910b that may be pre-loaded with reagents 1911 a, 1911 b. These componentsare similar to their counterparts in cartridge 1700, and will not bedescribed again in detail. As in the case of cartridge 1700, any desirednumber of reagent chambers, such as five or more, may be provided incartridge 1900.

Cartridge 1900 also includes a droplet generator, generally indicated at1912, which differs slightly from droplet generator 1712 of cartridge1700. Specifically, droplet generator 1912 includes two separate oilreservoirs 1914 a, 1914 b corresponding to, and separately connected to,the two different reagent chambers. Thus, oil reservoir 1914 a transfersoil to intersection point 1916 a, where the oil combines with aqueousfluid from reagent chamber 1910 a to form a first water-in-oil emulsionof sample/reagent droplets, and oil reservoir 1914 b transfers oil tointersection point 1916 b, where the oil combines with aqueous fluidfrom reagent chamber 1910 b to form a second water-in-oil emulsion ofsample/reagent droplets. Both emulsions then may be transferred to aninstrument 1900′ for thermocycling. In comparison to cartridge 1800,providing separate oil reservoirs and oil channels in the manner ofcartridge 1900 may reduce any chance of cross-contamination betweenreagents from the separate reagent chambers.

FIG. 50 is a schematic diagram depicting still another disposable samplepreparation cartridge, generally indicated at 2000, and suitable fluidicconnections between various components of the cartridge. Like cartridges1700 and 1900 depicted in FIGS. 42 and 49, respectively, cartridge 2000is configured to receive and prepare a target-containing sample for PCRthermocycling and amplification. Cartridge 2000 includes a samplechamber 2002, a waste chamber 2004, a first filter 2006, and a firstreservoir chamber 2008, which are similar to their counterparts incartridge 1700, and will not be described again in detail.

Cartridge 2000 also includes a second reservoir chamber 2009. Filter2006 is disposed between sample chamber 2002 and each of reservoirchambers 2008 and 2009, and serves to retain the target-containingsample in the sample chamber as fluid is transferred into and out of thesample chamber. As in the previously described exemplary cartridges,reconstitution and/or wash fluid will typically be transferred into thesample chamber from one of the reservoir chambers, and waste fluid willtypically be transferred out of the sample chamber into the wastechamber.

First and second reservoir chambers 2008 and 2009 are provided so thatthe sample in the sample chamber may be reconstituted and/or washedtwice. For example, a reconstitution solution may be transferred intothe sample chamber from reservoir chamber 2008, after which the samplemay be lysed as has been described previously. Waste fluid then may betransferred from the sample chamber into waste chamber 2004, while thetarget material is retained in the sample chamber. Next, a wash solutionmay be transferred into the sample chamber from reservoir chamber 2009,and waste fluid again may be transferred from the sample chamber intothe waste chamber. Providing two reservoir chambers and tworeconstitution/wash steps may result in a sample that containsrelatively few impurities and thus a relatively high fraction of targetmaterial.

A second filter 2007 is disposed between sample chamber 2002 and reagentchambers 2010 a, 2010 b. The reagent chambers may be pre-loaded withreagents 2011 a, 2011 b, and both the reagent chambers and the reagentsare similar to their previously described counterparts. Filter 2007 isconfigured to allow passage of target nucleotide material from thesample chamber to the reagent chambers, while preventing passage oflarger material, such as lysis beads or large waste material thatremains in the sample chamber after purification and lysis. As in thecase of cartridges 1700 and 1900, any desired number of reagentchambers, such as five or more, may be provided in cartridge 2000.

Alternatively, or in addition, to filter 2007, additional filters 2012a, 2012 b may be provided with reagent chambers 2010 a, 2010 b, andsimilar additional filters may be provided with each additional reagentchamber. These additional filters may serve a similar purpose as filter2007, i.e., preventing relatively large waste material, such as lysisbeads, from proceeding further through the cartridge. Providing both asecond filter 2007 and additional filters 2012 a, 2012 b may result in arelatively more pure sample/reagent mixture transferred from the reagentchambers toward a droplet generation portion of the cartridge.

Cartridge 2000 includes a droplet generator, generally indicated at2014, which is configured to generate a water-in-oil emulsioncorresponding to each reagent chamber. Unlike the previously describedcartridges, however, the oil for the emulsion is supplied by a relatedinstrument 2000′ rather than from within the cartridge. To describe theinteraction between the cartridge and the instrument, primed referencenumbers will be used to represent components of instrument 2000′,whereas unprimed reference numbers will continue to be used to referencecomponents of cartridge 2000.

To supply oil to cartridge 2000, an oil reservoir 2016′ withininstrument 2000′ transfers the oil along oil lines 2018 a, 2020 a, togenerate droplets corresponding to reagent chamber 2010 a. The oilintersects aqueous solution from reagent chamber 2010 a at anintersection region 2022 a, to generate droplets containing asample/reagent mixture that may be transferred into instrument 2000′ forthermocycling. Similarly, oil reservoir 2016′ supplies oil along lines2018 b, 2020 b to generate droplets corresponding to reagent chamber2010 b at an intersection region 2022 b, and oil reservoir 2016′ (oradditional reservoirs, not shown) may be configured to supply oil togenerate droplets corresponding to any desired number of additionalreagent chambers that are included in cartridge 2000.

Sample/reagent droplets generated at regions 2022 a, 2022 b, and at anyother additional droplet generation intersection regions of cartridge2000, all may be transferred through corresponding fluidic pathways 2024a, 2024 b (and so forth) to a multi-port valve 2026′ of instrument2000′. Valve 2026′ may, for example, be configured to receive dropletsfrom multiple fluidic input channels, and to transfer the droplets to athermocycling region of the instrument in any desired manner, such as incontrolled batches of one type of sample/reagent droplets at a time.

FIG. 51 is a schematic diagram depicting yet still another disposablesample preparation cartridge, generally indicated at 2100, and suitablefluidic connections between various components of the cartridge. Likethe previously described cartridges, cartridge 2100 is configured toreceive and prepare a target-containing sample for PCR thermocycling andamplification. Cartridge 2100 includes several of the features of theother cartridges, including a sample chamber 2102, a waste chamber 2104,a filter 2106, and reagent chambers 2110 a, 2110 b (plus any desirednumber of additional reagent chambers). These components are similar totheir previously described counterparts, and will not be described againin detail.

Cartridge 2100 is configured to be inserted into or otherwise interactwith a related PCR instrument 2100′, shown to the right of interfaceline L in FIG. 51. In this case, instrument 2100′ supplies substantiallyall of the working fluids, other than the sample or sample-containingfluid, to the cartridge. In other words, instrument 2100′ is configuredto prime cartridge 2100 with fluids. As in the case of the descriptionrelating to FIG. 50, primed reference numbers will be used in thedescription of FIG. 51 to represent components of instrument 2100′,whereas unprimed reference numbers will continue to be used to referencecomponents of cartridge 2100.

A reservoir pump 2112′ of instrument 2100′ may be equipped with aselector valve or similar mechanism to allow fluid to be selectivelytransferred from the reservoir pump through the various fluid channelsleading from the pump. After cartridge 2100 is placed in a secureposition within or adjacent to instrument 2100′, so that a substantiallyfluid tight seal is formed, the reservoir pump pumps fluid into fluidchannel 2114 toward waste chamber 2104, which is typically empty offluid when the cartridge is connected to the instrument. Reservoir pump2112′ continues pumping fluid into channel 2114 until the fluid fillschannel 2114 and proceeds through channel 2116 to fill filter 2106. Thereservoir pump then stops pumping fluid into channel 2114 and beginspumping fluid into channel 2118 a toward reagent chamber 2110 a,continuing until fluid fills channel 2118 a. During operation ofreservoir pump 2112′, a waste pump 2120′, which is fluidically connectedto reagent chamber 2110 a through a channel 2122 a, operates to drawaway air and any excess fluid.

Once fluid channels 2114, 2116, and 2118 a have been primed with fluid,reservoir pump 2112′ transfers a measured amount of fluid into fluidchannel 2124 between the reservoir pump and sample chamber 2102, to fillchannel 2124, channel 2126 a between the sample chamber and reagentchamber 2110 a, and channel 2122 a between reagent chamber 2110 a andwaste pump 2120′. Waste pump 2120′ operates to draw away air and fluidas channels 2124, 2126 a, and 2122 a are primed with fluid. Next,reservoir pump 2112′ transfers additional fluid through channel 2118 ato reagent chamber 2110 a, into channel 2130 a, through dropletgeneration region 2132 a, and into a multi-port valve 2134′ ofinstrument 2100′.

At this point, the fluid channels leading from reservoir pump 2112′ tosample chamber 2102, waste chamber 2104, and reagent chamber 2110 a, andfrom reagent chamber 2110 a to multi-port valve 2134′, have all beenprimed with fluid. Reservoir pump 2112′ may then be used to prime thefluid channels associated with any additional reagent chambers. Forexample, reservoir pump 2112′ may transfer a measured amount of fluidthrough channel 2124 to fill channel 2126 b between the sample chamberand reagent chamber 2110 b, and channel 2122 b between reagent chamber2110 b and waste pump 2120′, while waste pump 2120′ operates to drawaway air and fluid. Reservoir pump 2112′ then may transfer fluid throughchannel 2128 b directly to reagent chamber 2110 b, into channel 2130 b,through droplet generation region 2132 b, and into multi-port valve2134′. In a similar manner, reservoir pump 2112′ (or in some cases,additional reservoir pumps) can be used to prime the fluid channelsassociated with any desired number of reagent chambers.

Once the channels of cartridge 2100 have been primed to a desireddegree, a sample or sample-containing fluid may be placed in the samplechamber, and all of the previously described steps of purification,concentration, lysing, reagent combination, and/or droplet generationmay be performed as described previously with respect to other cartridgeembodiments. However, one additional distinction between cartridge 2100and the previously described cartridges is that cartridge 2100 does notinclude an oil reservoir to supply oil for droplet generation. Rather,an oil reservoir 2140′ is included in instrument 2100′. Oil reservoir2140′ is configured to supply oil through lines 2142 a and 2144 a todroplet generation region 2132 a, and through lines 2142 b and 2144 b todroplet generation region 2132 b. The oil reservoir can be configured tosupply oil to any desired number of additional droplet generationregions, corresponding to additional reagent reservoirs beyond the twodepicted in FIG. 51. After sample/reagent droplets are generated, theymay be transferred to multi-port valve 2134′, which is configured totransfer the droplets to a thermocycling portion of instrument 2100′ forPCR amplification.

F. Example 6: Disposable Sample Cartridge 4

This example describes aspects of yet another alternative disposablesample preparation cartridge; see FIGS. 52 and 53.

FIG. 52 is an isometric view of an interior portion of the exemplarycartridge, generally indicated at 2150. Cartridge 2150 is configured tointerface with an instrument (not shown), so that prepared samples canbe transferred to the instrument, generally in the form of awater-in-oil emulsion, for PCR amplification and analysis. In additionto the interior portion depicted in FIG. 52, cartridge 2150 also mayinclude a suitable exterior housing (not shown) disposed around some orthe entirety of the interior portion. The exterior housing may beconfigured to protect the interior portion and may be shaped tofacilitate storage and/or transportation of multiple cartridges.

Cartridge 2150 includes an upper body portion 2152, plus variousplungers and connectors that will be described in more detail below.Body portion 2152 may be unitarily constructed, for example, byinjection molding a thermoplastic or other similar material. A second,lower body portion (not shown) may be included in cartridge 2150 andconnected to the upper body portion by heat sealing, gluing, orotherwise fastening the two body portions together, but this lower bodyportion is simply a substantially planar, featureless sheet of materialand therefore will not be described further. Restricting the significantfeatures within a unitarily constructed cartridge body portion, such asupper body portion 2152, may have advantages in cost, simplicity,structural integrity, and/or improved functionality compared to atwo-piece construction where both pieces include features used for fluidmanipulation and transfer, as shown and described (for example) withreference to FIGS. 43-44 above.

Body portion 2152 of cartridge 2150 includes a sample chamber 2154configured to receive a sample that potentially contains a targetnucleic acid sequence, a reservoir chamber 2156 configured to supply awash and/or a reconstitution solution, a waste chamber 2158 fluidicallyconnected to the sample chamber and configured to receive wastematerial, and various reagent chambers 2160 a, 2160 b, 2160 c, 2160 d,2160 e each fluidically connected to the sample chamber and configuredto receive sample-containing fluid and to combine the sample-containingfluid with a reagent mixture prior to PCR thermocycling. In addition,body portion 2152 of cartridge 2150 includes droplet chambers 2161 a,2161 b, 2161 c, 2161 d, 2161 e, each of which is configured to receivean emulsion of water-in-oil, sample-containing droplets including thesample/reagent mixture contained in the corresponding reagent chamber.As described previously, any desired number of reagent chambers (andcorresponding droplet chambers) may be included in a cartridge. Thesample chamber, reservoir chamber, waste chamber, and reagent chambersare substantially similar in both structure and function to theircounterparts in cartridge 1720 of FIG. 43, including any appropriatefilters, stirring elements, and the like, and accordingly will not bedescribed in detail again.

Body portion 2152 also includes an oil input chamber 2162, an oil outletchamber 2164, and a primer outlet chamber 2166. Oil input chamber 2162is configured to hold and transfer oil that will be used to producesample-containing droplets in a water-in-oil emulsion, in a mannerdescribed below in more detail. Oil outlet chamber 2164 is configured toreceive oil that has been transferred out of the oil input chamber, butthat has not been utilized in the water-in-oil emulsion ofsample-containing droplets. The excess oil received in oil outletchamber 2164 may be either discarded or recycled (i.e., redirected tothe oil input chamber). Primer outlet chamber 2166 is configured toreceive one or more priming fluids during an initial cartridge primingstep, in a manner that will be described in more detail below.

In addition to upper body portion 2152, cartridge 2150 also includes afluid manipulation portion, generally indicated at 2168. The fluidmanipulation portion of the cartridge includes a sample chamber plunger2170 and various reagent chamber plungers 2172 a, 2172 b, 2172 c, 2172d, 2172 e. The plungers are configured to move up and down within theirrespective chambers, to cause fluid to be transferred into and out ofthe chambers in a desired fashion. Fluid manipulation portion 2168 ofthe cartridge also includes a plurality of substantially similarcapillary connectors 2174, and a plurality of substantially similarcapillaries 2176. The capillary connectors are configured to transferfluid to and/or from the corresponding chamber to the correspondingcapillary, which is configured to interface with an associatedthermocycling instrument.

FIG. 53 is a bottom view of upper body portion 2152, illustrating anetwork of fluid channels forming the fluid connections between variousportions of the cartridge. As noted above, a lower body portion (notshown) of cartridge 2150 will generally be disposed flush against thebottom surface of upper body portion 2152, to form a fluid tight seal sothat fluid is only able to travel between portions of the cartridgethrough the various fluid channels shown in FIG. 53. Thus, the networkof fluid channels is defined by a lower surface of the upper bodyportion and an upper surface of the lower body portion, although theupper surface of the lower body portion is in this example asubstantially planar surface, so that the fluid channels are formedentirely in the upper body portion of the cartridge.

Specifically, a fluid channel 2178 is configured to transferreconstitution/wash and/or priming fluid into sample chamber 2154 fromreservoir chamber 2156, and another fluid channel 2180 is configured totransfer waste fluid out of sample chamber 2154 and into waste chamber2158. Yet another fluid channel 2182 is configured to transfersample-containing fluid from sample chamber 2154 into reagent chambers2160 a, 2160 b, 2160 c, 2160 d, 2160 e, and also to transfer primingfluid from sample chamber 2154 into primer outlet chamber 2166. Yetanother fluid channel 2184 is configured to transfer oil from oil inputchamber 2162 to a plurality of droplet generation regions 2186 a, 2186b, 2186 c, 2186 d, 2186 e. The droplet generation regions are eachfluidically connected to one of the reagent chambers and each configuredto receive sample/reagent mixture fluid from one of the reagent chambersand to combine the sample/reagent mixture fluid with a background fluidto form an emulsion of sample-containing droplets. A plurality of fluidchannels 2188 a, 2188 b, 2188 c, 2188 d, 2188 e are configured totransport the generated droplets from their respective dropletgeneration regions to corresponding droplet chambers 2161 a, 2161 b,2161 c, 2161 d, 2161.

Typically, cartridge 2150 will be primed with fluid(s) supplied by arelated instrument. For instance, when a fluid connection has beenestablished between the cartridge and the instrument, priming fluid suchas oil, water, or any other substantially incompressible fluid may betransferred from the instrument, through the appropriate capillary andcapillary connector, and into reservoir chamber 2156. The priming fluidthen may be transferred from the reservoir chamber, through fluidchannel 2178, and into sample chamber 2154. From the sample chamber, thepriming fluid may be transferred through fluid channel 2182 and intoprimer outlet chamber 2166 and/or the reagent chambers. Similarly, oilor some other priming fluid may be transferred from the instrument intooil input chamber 2162, through fluid channel 2184, and into oil outletchamber 2164 and/or the droplet generation chambers. In this manner,desired priming fluids can be used to prime any desired subset of thefluid chambers and channels of cartridge 2150.

Plungers 2170, 2172 a, 2172 b, 2172 c, 2172 d, and 2172 e (and any otherplungers contemplated by the present disclosure) each may be configuredboth to direct fluids as desired through particular fluid channels, andalso to selectively allow or prevent fluid flow in and out of variouschambers. In other words, each plunger may be configured to operate as avalve in addition to operating as a plunger, by selectively opening orclosing the entrance to one or more particular fluid channels. Forexample, when reagent plungers 2172 a, 2172 b, 2172 c, 2172 d, and 2172e are in their most downward positions (minimizing the volumes of thereagent chambers), the plungers may be configured to block fluidconnection between fluid channel 2182 and fluid channel 2184 (see FIG.53), so that channel 2182 can be primed with fluid independently ofchannel 2184. In a similar manner, the plungers of any cartridge can beused as valves, to prevent or allow fluid flow between various portionsof the cartridge.

Disposable cartridge 2150 of FIGS. 52 and 53 is just one example of adisposable cartridge that is configured to be primed with fluid suppliedby an associated instrument. The present disclosure contemplates otherdisposable cartridges that may be substantially similar except for thedisposition of various chambers and/or variations in how fluids arerouted between the various chambers, or between the chambers and theinstrument. For example, the waste chamber and/or the reservoir chambermay be disposed on the instrument rather than on the cartridge as inFIGS. 52 and 53. A plurality of oil input chambers may be provided, witheach chamber supplying oil to a single droplet generation region ratherthan one chamber supplying oil to multiple regions as in FIGS. 52 and53. The droplet generation regions may take any of the various formsdescribed previously with respect to FIGS. 48A-48F, such as a crossconfiguration instead of a single T configuration as in FIGS. 52 and 53.Excess oil or priming fluid may either be discarded as in FIGS. 52 and53, recycled, or routed through the droplet generator outlet(s).Droplets may be routed either through multiple outlets as in FIGS. 52and 53 or through a single, common outlet. Virtually any combination ofthe above variations may be adopted, resulting in a modified system thatmay be most appropriate for a particular application.

G. Example 7: Selected Embodiments

This subsection describes additional aspects of sample preparation andsample cartridges, in accordance with aspects of the present disclosure,presented without limitation as a series of numbered sentences.

1. A method of target molecule amplification, comprising (A) purifying afluid sample; (B) lysing the sample; (C) combining the sample with areagent mixture; (D) generating droplets of the sample in an emulsion;and (E) transferring the emulsion to a thermocycling instrument; whereinthe steps of purifying, lysing, combining, and generating are allperformed within a disposable, single-use cartridge.

2. The method of paragraph 1, further comprising extracting the samplefrom a sample collector within the disposable cartridge.

3. The method of paragraph 1, further comprising concentrating thesample within the disposable cartridge.

4. The method of paragraph 1, wherein purifying includes purifying priorto lysing by retaining target material within the sample while removingwaste material smaller than the target material.

5. The method of paragraph 1, wherein purifying includes purifying afterlysing by retaining target material within the sample while removingwaste material larger than the target material.

6. A single-use sample preparation cartridge, comprising a first bodyportion and a second body portion, wherein the first body portionincludes (A) a sample chamber configured to receive a sample; (B) areservoir chamber fluidically connected to the sample chamber andconfigured to supply a reconstitution fluid to the sample chamber; (C) awaste chamber fluidically connected to the sample chamber and configuredto receive waste fluid from the sample chamber; (D) a plurality ofreagent chambers each fluidically connected to the sample chamber andeach configured to receive sample-containing fluid from the samplechamber and to combine the sample-containing fluid with a reagentmixture; and (E) a plurality of droplet generation regions, eachfluidically connected to one of the reagent chambers and each configuredto receive sample/reagent mixture fluid from one of the reagent chambersand to combine the sample/reagent mixture fluid with a background fluidto form an emulsion of sample-containing droplets; and wherein thesample chamber, the reservoir chamber, the waste chamber, the reagentchambers, and the droplet generation regions are fluidically connectedto each other by a network of fluid channels defined by a lower surfaceof the first body portion and an upper surface of the second bodyportion.

7. The cartridge of paragraph 6, wherein the fluid channels are formedentirely in the first body portion, and wherein the upper surface of thesecond body portion is a substantially planar surface.

8. The cartridge of paragraph 6, wherein the background fluid is oil,and further comprising an oil input chamber configured to receive oil tobe transferred to the droplet generation regions.

9. The cartridge of paragraph 8, further comprising an oil outletchamber configured to receive oil that has been transferred out of theoil input chamber, but that has not been utilized in one of theemulsions.

10. The cartridge of paragraph 6, further comprising a plurality ofdroplet chambers each configured to receive one of the generatedemulsions.

11. The cartridge of paragraph 6, further comprising a fluidmanipulation portion including a plurality of plungers configured tocause fluid to be transferred into and out of the chambers.

12. The cartridge of paragraph 11, wherein the fluid manipulationportion further includes a plurality of connectors configured totransfer fluid between at least one chamber of the cartridge and theinstrument.

13. The cartridge of paragraph 11, wherein each plunger is configured toact as a valve by selectively closing an entrance to at least one of thefluid channels when in its most downward position.

14. The cartridge of paragraph 11, wherein the sample chamber includesan agitation element configured to be agitated by magnetic forces.

15. The cartridge of paragraph 11, wherein the reagent chambers arefluidically connected to the sample chamber in parallel.

16. The cartridge of paragraph 11, wherein the background fluid is oil,and further comprising at least one oil reservoir fluidically connectedto at least one of the reagent chambers and configured to supply the oilused to form the corresponding emulsion.

17. The cartridge of paragraph 16, wherein the at least one oilreservoir includes one oil reservoir corresponding to each reagentchamber and configured to supply the oil used to form the correspondingemulsion.

18. A microfluidic device having integrated lysing, separating, reagentmixing and microdroplet generating regions for extracting nucleic acidfrom a sample and for formation of microdroplets, comprising (A) alysing region for lysing a cell or microorganism to release the nucleicacid; (B) a separating region for separating the nucleic acid from otherparts of the cell or microorganism, wherein the separating region isconnected to the lysing region; (C) a reagent mixture region for mixingthe nucleic acid with at least one reagent; wherein the reagent mixtureregion is connected to the separating region; and (D) a dropletgenerating region comprising a sample inlet end, an immiscible fluid,and an outlet end, wherein the droplet generating region is connected tothe reagent mixture region.

IV. DROPLET GENERATOR

This Section describes exemplary droplet generators, for example, foruse in droplet-based assays.

It may be desirable, in systems such as DNA amplification systems, amongothers, to generate sample-containing droplets using a partially orcompletely disposable apparatus. This may be accomplished by adisposable cartridge configured to generate droplets as part of a seriesof sample preparation steps that also may include lysing, purification,and concentration, among others. However, in other cases, it may bedesirable to provide a partially or completely disposable apparatusconfigured to perform droplet generation without performing substantialadditional sample preparation steps. This may be desirable, for example,when the DNA amplification system is configured to analyze samples thatare typically prepared at another location or by a practitioner. Underthese circumstances, a dedicated droplet generation system may be thesimplest and most economical solution.

FIG. 54 schematically illustrates a droplet generation system, generallyindicated at 2200. System 2200 includes a droplet generator 2202 and afluid reservoir 2204. Droplet generator 2202 is configured to generatesample-containing droplets, typically in the form of a water-in-oilemulsion, and to transport the generated droplets to a desired locationsuch as a storage location or a thermocycling instrument. Fluidreservoir 2204 is configured to store and/or receive the fluids thatwill be used to form the emulsion, typically a background fluid such asoil and a foreground fluid such as an aqueous solution containing a DNAsample and a reagent mixture.

To generate an emulsion of droplets, droplet generator 2202 willtypically be at least partially disposed within fluid reservoir 2204, asFIG. 54 indicates. To transport droplets away from reservoir 2204,droplet generator 2202 will typically either be physically removablefrom the reservoir, or will include suitable fluid connections,schematically indicated at 2206, configured to receive droplets from thedroplet generator and to transfer them to another desired location. Whendroplet generator 2202 is configured to be removable from reservoir2204, one or both of the droplet generator and the reservoir may bedisposable. Disposing of any portions of the system that have come intodirect contact with a sample may, for example, help to avoid thepossibility of cross-contamination between multiple samples.

Many configurations of droplet generators and fluid reservoirs may besuitable as components of a droplet generation system such as system2200. For example, suitable droplet generators include butted tubes,tubes drilled with intersecting channels, tubes partially or completelyinserted inside other tubes, and tubes having multiple apertures, amongothers, where “tubes” means elongate hollow structures of anycross-sectional shape. Suitable fluid reservoirs include pipette tips,spin columns, wells (either individual or in a plate array), tubes, andsyringes, among others. The following examples describe specificexemplary droplet generators and fluid reservoirs; see FIGS. 55-71.Additional pertinent disclosure may be found in the U.S. provisionalpatent applications listed above under Cross-References and incorporatedherein by reference, particularly Ser. No. 61/277,204, filed Sep. 21,2009.

A. Example 1

FIGS. 55 and 56 depict exemplary cross-type droplet generators.

FIG. 55 schematically depicts a first exemplary cross-type dropletgenerator, generally indicated at 2210, in the form of a pair of buttedtubes. The term “cross-type droplet generator” indicates that abackground emulsion fluid (typically oil) travels inward from twosubstantially opposite directions to intersect a foreground emulsionfluid (typically an aqueous fluid) traveling at right angles to thedirection of travel of the background fluid, to form an emulsion thatmoves along the original direction of travel of the foreground fluid.Thus, the directions of travel of the incoming background fluid, theincoming foreground fluid, and the outgoing emulsion form a cross.

Accordingly, droplet generator 2210 includes two complementary sectionsof hollow fluidic tubing 2212, 2214, separated by a small distance D.Tubing sections 2212, 2214 may be constructed from a single continuoushollow tube that has been cut and separated, in which case the tubingsections will have substantially equal outer and inner diameters.Alternatively, tubing sections 2212, 2214 may be constructed separatelyand then disposed appropriately within droplet generator 2210, in whichcase the tubing sections may have substantially different outer and/orinner diameters.

Tubing sections 2212, 2214 are disposed at least partially within an oilchannel 2216. Oil channel 2216 will typically be a portion of a fluidreservoir configured to supply fluids, including oil and/orsample-containing aqueous fluid, to droplet generator 2210. Variousexemplary fluid reservoirs are described in Example 2 below. Oil channel2216 may take various forms, such as a cylindrical channel formed withina tube, a rectangular channel formed between substantially planarchannel walls, or simply a fluid flow path within a surroundingreservoir of fluid, among others. Tubing sections 2212, 2214 may beformed integrally with oil channel 2216, or the tubing sections may beinserted into one or more apertures of the oil channel in asubstantially fluid tight manner.

Tubing section 2212 includes a hollow inner portion forming an incomingfluid channel 2218, and tubing section 2214 includes a hollow innerportion forming an outgoing fluid channel 2220. Incoming fluid channel2218 is configured to transport sample-containing fluid from a fluidsource such as a surrounding fluid reservoir or a reagent chamber intooil channel 2216, and may be pressurized relative to the oil channel tofacilitate that transfer. To generate sample-containing droplets, oil inoil channel 2216 and sample-containing fluid in incoming fluid channel2218 each may be pressurized relative to outgoing fluid channel 2220,tending to draw both oil and sample-containing fluid toward an inletaperture 2222 of the outgoing fluid channel. As the sample-containingfluid exits an outlet aperture 2224 of incoming fluid channel 2218,aqueous droplets of sample-containing fluid may be formed in an oilbackground, resulting in a water-in-oil emulsion of droplets enteringthe outgoing fluid channel.

One of tubing sections 2212, 2214 may be fixed within a surroundingfluid reservoir, whereas the other section may be removable from thesurrounding reservoir. In such cases, tubing section 2212 will typicallybe fixed in place, whereas tubing section 2214 will typically beremovable, and may be configured to be selectively placed into positionat a known, desired distance from tubing section 2214. For example,tubing section 2214 may represent the tip of a syringe, pipette, or thelike, which may be inserted into a reservoir containing oil channel 2216and used to create and store sample-containing droplets by applyingsuction to draw an emulsion of sample-containing droplets into inletaperture 2222 of the outgoing fluid channel. Tubing section 2214 thenmay be removed from the fluid reservoir, and the emulsion transferred toanother desired location such as a thermocycling instrument.

FIG. 56 depicts a second exemplary cross-type droplet generator,generally indicated at 2230. Droplet generator 2230 is constructed froma single section of fluidic tubing, through which two perpendicular andintersecting fluid channels 2232 and 2234 are formed. Droplet generator2230 may be temporarily or permanently disposed within a fluid reservoir(not shown) configured to hold fluids used to form an emulsion ofsample-containing droplets, such as a background oil and a foregroundsample-containing aqueous solution. A distal aperture 2236 of fluidchannel 2232 is configured to receive and transport thesample-containing solution, and intermediate apertures 2238, 2240 offluid channel 2234 is configured to receive and transport the backgroundoil.

At an intersection region generally indicated at 2242, sample-containingfluid traveling through channel 2232 intersects with oil travelingthrough channel 2234, and a water-in-oil emulsion of sample-containingdroplets is generated. This emulsion then continues to travel throughchannel 2232 along the original direction of travel of thesample-containing fluid (from left to right in FIG. 56). The emulsionthen may be transferred to a storage location and/or to a thermocyclinginstrument as is desired. In some cases, droplet generator 2230 may bethe tip of a removable and/or disposable component such as a syringe orpipette, or alternatively, droplet generator 2230 may represent thedistal portion of a fixed, nondisposable component that is configured totransport a droplet emulsion away from a fluid reservoir to a desiredlocation.

B. Example 2

FIGS. 57 and 58 depict exemplary flow-focus droplet generators.

FIG. 57 depicts a first exemplary flow-focus droplet generator,generally indicated at 2250. The term “flow-focus droplet generator”indicates that droplets are generated when a background fluid is focusedby the local geometry of its surroundings toward an intersection regionwhere it intersects a foreground, sample-containing fluid. An emulsionof sample-containing droplets is then formed. Unlike in a cross-typedroplet generator, the background and foreground fluids in a flow-focusdroplet generator need not intersect at substantially right angles, asindicated in FIG. 57.

Flow-focus droplet generator 2250 includes a fluid input channel 2252, adroplet output channel 2254, and an oil reservoir 2256. Fluid inputchannel 2252 is configured to transport sample-containing fluid toward afluid intersection region generally indicated at 2258. As FIG. 57depicts, fluid input channel 2252 may be substantially cylindrical withan elongate tapered tip 2260 configured to produce fluid droplets of adesired size, although variations such as a non-tapered tip also may besuitable. Droplet output channel 2254 also may be substantiallycylindrical or have any other desired shape suitable for directing thebackground oil toward intersection region 2258 in conjunction with tip2260, as described below. Oil reservoir 2256 is configured to receiveand/or store oil or any other suitable emulsion background fluid.

To generate droplets, a pressure differential is created to draw fluidfrom both input channel 2252 and oil reservoir 2256 into output channel2254. Due to the geometry of the input channel, the output channel, andthe reservoir, oil from the reservoir forms a fluid path that is focusedtoward intersection region 2258 with a component of fluid velocityparallel to the direction of travel of the sample-containing fluidwithin the fluid input channel, as indicated by arrows 2262 in FIG. 57.An emulsion of sample-containing droplets in an oil background is formedand travels away from intersection region 2258 within fluid outputchannel 2254, in substantially the same direction of motion as thedirection of motion of the sample-containing fluid within fluid inputchannel 2252.

Output channel 2254 either may be fixed within oil reservoir 2256, inwhich case it will be configured to transfer the generated water-in-oilemulsion out of the oil reservoir to another desired location such as astorage location or a thermocycling instrument. Alternatively, outputchannel 2254 may be part of a removable and/or disposable component suchas the tip of a syringe or a pipette, in which case it may be removedonce a desired amount of emulsion has been generated. The emulsion thenmay be physically transported, in bulk, to another desired location.

FIG. 58 depicts a second flow-focus droplet generator, generallyindicated at 2280. Droplet generator 2280 is similar to dropletgenerator 2250 of FIG. 57, except that droplet generator 2280 does notinclude a separate sample-containing fluid input channel. Instead,droplet generator 2280 includes only a droplet output channel 2282 and afluid reservoir 2284. In this case, however, fluid reservoir 2284 isconfigured to receive and/or store both sample-containing fluid and asuitable emulsion background fluid such as oil. As in the embodiment ofFIG. 57, the droplet output channel may be part of a removable and/ordisposable component.

To generate droplets with droplet generator 2280, a pressuredifferential is created to draw fluid into output channel 2282. Againdue to the local geometry of the area near a fluid intersection region2286, oil from the reservoir forms a fluid path that is focused towardintersection region 2286, as indicated by arrows 2288. In addition,sample-containing fluid is drawn toward intersection region 2286, wherethe meniscus at the boundary between the sample-containing fluid and theoil forms a necking region 2290 adjacent to the intersection region. Inthe necking region, the meniscus is periodically deformed into anelongate “neck,” at which point a discrete droplet is separated from themeniscus. An emulsion of sample-containing droplets in an oil backgroundis thus formed as droplets are generated one at a time in the neckingregion.

C. Example 3

FIGS. 59 and 60 depict yet another cross-type droplet generator,generally indicated at 2300. Droplet generator 2300 includes adisposable sample-containing portion 2302, and a nondisposable dropletoutlet portion 2304. Sample-containing portion 2302 may be configured tobe a single-use, disposable component, and accordingly may beconstructed of a relatively inexpensive material such as aninjection-molded thermoplastic. FIG. 59 depicts droplet generator 2300with sample-containing portion 2302 and droplet outlet portion 2304substantially separated from each other and thus not in a positionsuitable for producing sample-containing droplets. FIG. 60 depictsdroplet generator 2300 with sample-containing portion 2302 and dropletoutlet portion 2304 disposed in close proximity to each other, inposition for producing sample-containing droplets as described below.

Sample-containing portion 2302 of droplet generator 2300 includes asample reservoir 2306 and a sample fluid channel 2308. The samplereservoir may be configured to receive sample-containing fluid throughany suitable fluid input mechanism such as fluidic tubing (not shown),manual insertion of sample-containing fluid by a practitioner, orautomatic insertion of sample-containing fluid by a machine. Samplefluid channel 2308 is configured to transport fluid from the samplereservoir toward a fluid outlet aperture 2310, which is configured toemit droplets of sample-containing fluid that have passed through thesample fluid channel from the sample reservoir. Sample-containingportion 2302, sample reservoir 2306, and sample fluid channel 2308depicted in the cross-sectional view of FIGS. 59-60 are allsubstantially cylindrical, although other shapes may be suitable.

Droplet outlet portion 2304 of droplet generator 2300 includes anemulsion outlet channel 2312, which is configured to transport anemulsion of sample-containing droplets toward a desired location such asa storage chamber or a thermocycling instrument (not shown). Dropletoutlet portion 2304 also includes an oil channel 2314, which is definedby upper and lower channel walls 2316, 2318 of the outlet portion. Oilchannel 2314 may take the form of an elongate groove, a cylindrical (oralternately shaped) substantially planar reservoir, or any other desiredform suitable for facilitating the transfer of oil toward droplet outletchannel 2312.

A substantially cylindrical aperture 2320 is formed in upper channelwall 2316 of the droplet outlet portion, and is configured to receive acomplementary cylindrical lower part 2322 of sample-containing portion2302. A fluid tight sealing ring 2324, such as an o-ring, may beprovided to help form a substantially fluid tight seal betweensample-containing portion 2302 and droplet outlet portion 2304 when thetwo portions are assembled together. A cylindrical groove may be formedin the exterior surface of sample-containing portion 2302 to retain theo-ring in a desired position, and another similar groove may be providedwithin aperture 2320. Aligning the o-ring within these grooves may helpa user to locate the correct mounting position of the sample-containingportion within cylindrical aperture 2320. Alternatively or in addition,various locating pins or other similar protrusions (not shown) may beprovided and attached to one or both of the sample-containing portionand the droplet outlet portion, to stop those portions at a desiredseparation distance from each other when the sample-containing portionis mounted to the droplet outlet portion.

FIG. 60 shows the two main portions of droplet generator 2300 assembledtogether and droplets being formed. Oil travels within oil channel 2314,inward toward droplet outlet channel 2312, as indicated by arrows 2330.At the same time, sample-containing fluid travels downward throughsample fluid channel 2308 to intersect the oil at an intersection regiongenerally indicated at 2332. At intersection region 2332, an emulsion ofwater-in-oil droplets is produced and passes into droplet outlet channel2312. All of these fluid motions are typically caused by negativepressure introduced at a distal end of the droplet outlet channel. Thegenerated emulsion may pass through the outlet channel and into astorage chamber, a transport chamber, or directly to a thermocyclinginstrument. In summary, when the droplet outlet portion and thesample-containing portion of droplet generator 2300 are assembledtogether, a substantially fluid tight seal is formed between the dropletoutlet portion and the sample-containing portion, and droplets emittedby the fluid outlet aperture intersect oil traveling in the oil channelto produce an emulsion of water-in-oil droplets that passes into theemulsion outlet channel.

When oil channel 2314 takes the form of an elongate groove, the oil andsample-containing fluid intersect and produce droplets with the variousfluid velocities forming a cross shape, as described previously. If oilchannel 2314 takes the form of an extended planar channel or reservoir,the oil within the channel may approach droplet outlet channel 2312radially from many different directions, each of which is substantiallyperpendicular to both the sample fluid channel and the droplet outletchannel. Accordingly, such a configuration still may be thought of as across-type droplet generator.

Sample-containing portion 2302 of droplet generator 2300 may bedisposable, as mentioned previously. Thus, after an emulsion is createdand transported to a desired location, sample-containing portion 2302may be removed from aperture 2320 and discarded. Anothersample-containing portion then may be placed into aperture 2320 and usedto create another emulsion, using either the same or a differentsample/reagent mixture. The internal surfaces of droplet outlet portion2304, including the walls of outlet channel 2312 and channel walls 2316,2318, all may be coated with a hydrophobic coating and/or washed withone or more rinse solutions, to reduce the possibility of crosscontamination from one sample/reagent solution to another.

D. Example 4

FIGS. 61-63 depict exemplary droplet generation systems generallyconfigured to generate an emulsion of relatively less dense fluiddroplets in a background of relatively more dense fluid.

FIG. 61 depicts a first such droplet generation system, generallyindicated at 2340, including both a droplet generator 2342 and a fluidreservoir 2344. Droplet generator 2342 includes a substantiallycylindrical emulsion chamber 2346 and an elongate tip 2348, althoughother emulsion chamber and tip shapes are possible. The tip of thedroplet generator is configured to be at least partially inserted intothe fluid reservoir. Droplet generator 2342 also includes an interfaceportion 2350, which is configured to join emulsion chamber 2346 to abody portion of the droplet generator (not shown). The body portion ofthe droplet generator may, for example, be configured to be grasped by auser, and may include a pressure mechanism such as a pipettor bulb, asyringe plunger or the like, to effect pressure changes within thedroplet generator.

Tip 2348 of the droplet generator is depicted as cylindrical, i.e., ashaving a circular cross-section, but the cross-section of the tip (andof the emulsion chamber) can take many other shapes, such asrectangular, square, or oval. The tip includes both a distal endaperture 2352 configured to receive a background fluid such as oil, anda side aperture 2354 configured to receive a foreground fluid such as anaqueous sample/reagent mixture. In some cases, distal aperture 2352 willbe formed simply by leaving the distal end of tip 2348 open, andaccordingly will have the same shape as a cross-section of the tip.However, the distal aperture may be given any desired shape tofacilitate a desired flow rate of background fluid into the aperture.Side aperture 2354 may be formed in various shapes, such as circular,square, rectangular, star-shaped, oval, or triangular, among others. Theshape of side aperture 2354 may be selected based on a desired flow rateand/or flow pattern of fluid passing through the side aperture.

Fluid reservoir 2344 is depicted substantially as a parabaloid, butvirtually any three dimensional container that is closed at one end andopen at another may form a suitable reservoir. The fluid reservoir may,for example, be one of many reservoirs disposed in an array on a chip ora microplate, or it may be a single freestanding reservoir such as anindividual well, a test tube, a pipette body, or a spin column chamber,among others. Regardless of its precise shape, reservoir 2344 isconfigured to hold both a background emulsion fluid and a foregroundemulsion fluid, which will be used in conjunction with droplet generator2342 to form an emulsion of sample-containing droplets as describedbelow.

FIG. 62 shows a magnified view of a portion of the droplet generationsystem of FIG. 61, illustrating how an emulsion of sample-containingdroplets can be generated by the system. As shown, reservoir 2344 isconfigured to hold both a background emulsion fluid 2356 (such as oil)and a foreground emulsion fluid 2358 (such as an aqueous sample/reagentmixture). In system 2340, background fluid 2356 has a different andgreater density than foreground fluid 2358, and thus is disposed at thebottom portion of reservoir 2344, with the foreground fluid disposed ina layer above the background fluid. Accordingly, distal aperture 2352 ofdroplet generator 2342 is in contact with the background fluid, whereas,side aperture 2354 of droplet generator 2342 is in contact with theforeground fluid. In other words, the distal aperture is configured tobe in contact with background fluid held by the reservoir and the sideaperture is configured to be in contact with foreground fluid held bythe reservoir when the reservoir contains background and foregroundfluids and the elongate tip is inserted into the reservoir.

To generate an emulsion of foreground-in-background fluid droplets, anegative or upward pressure is applied to an interior fluid channel 2360of droplet generator 2342. This pressure may be applied by any suitablemechanism such as a manual or motor-driven plunger, a bulb, or a pump,among others. In any case, the applied pressure causes background fluid2356 to flow into distal aperture 2352 of droplet generator 2342, andalso causes foreground fluid to flow into side aperture 2354 of dropletgenerator 2342. Accordingly, foreground fluid flowing into the sideaperture intersects with a stream of background fluid that enters thetip through the distal aperture, to form an emulsion of foreground fluiddroplets 2362 in background fluid in the vicinity of the side aperture.An emulsion of droplets 2362 in background fluid then proceeds upchannel 2360, where it is received in emulsion chamber 2346. Theemulsion then may be stored and/or transported to another location suchas to a thermocycling instrument for DNA amplification, as describedpreviously. Because the directions of the incoming background fluidvelocity, the incoming foreground fluid velocity, and the outgoingemulsion velocity form the shape of a “T,” the system shown in FIGS.61-62 may be described as a “single T” droplet generator configuration.

FIG. 63 shows a magnified end portion of another droplet generationsystem, generally indicated at 2380, which is similar to system 2340 ofFIGS. 61 and 62. Specifically, system 2380 includes a droplet generator2382 and a fluid reservoir 2384 having all the same features as thecorresponding parts of system 2340, except that tip 2385 of dropletgenerator 2382 includes a distal end aperture 2386 and two distinct sideapertures 2388, 2390, all of which provide fluid access to a fluidchannel 2392 within the tip of the droplet generator. Accordingly, whenupward pressure is applied to fluid channel 2392, a background fluid2394 flows into distal aperture 2386, and a foreground fluid 2396 flowsinto both side apertures 2388, 2390. This may result in a greater numberand/or a different distribution of droplets being produced in anemulsion, relative to systems with just a single side aperture.

Because of the directions of the various fluid velocities in thevicinity of side apertures 2388, 2390, system 2380 may be characterizedas a “double T” droplet generator configuration. This configuration maybe generalized in various ways. For example, a pair of side aperturesmay be disposed at the same longitudinal position along the tip of adroplet generator, rather than longitudinally offset as depicted in FIG.63. Furthermore, any desired number of side apertures, such as three ormore, may be disposed along the length of a droplet generator, some ofwhich may be longitudinally aligned while others are longitudinallyoffset. Because the fluid velocities form a “T” at each side aperture,such generalized configurations naturally may be characterized as“multi-T” droplet generation systems. The number, location, size andshape of the various side apertures in a multi-T system typically willbe selected based on the desired properties of the resulting emulsion.

E. Example 5

FIGS. 64-66 depict droplet generation systems generally configured togenerate an emulsion of relatively more dense fluid droplets in abackground of relatively less dense fluid. In contrast, FIGS. 61-63, inthe previous example, depicted droplet generation systems generallyconfigured to generate an emulsion of relatively less dense fluiddroplets in a background of relatively more dense fluid.

FIG. 64 depicts a magnified end portion of a first such dropletgeneration system, generally indicated at 2400. System 2400 includes adroplet generator 2402 and a fluid reservoir 2404. Fluid reservoir 2404is substantially similar to reservoir 2344 depicted in FIGS. 61-62,including all of the possible variations in its structure and shape, andaccordingly will not be described again in detail. A foreground fluid2406 of relatively high density is disposed at the bottom of reservoir2404, and a background fluid 2408 of relatively low density is disposedabove the foreground fluid within the reservoir.

Droplet generator 2402 includes a tip 2410, the interior of which formsa fluid channel 2412, a distal aperture 2414, and a side aperture 2416.However, tip 2410 of droplet generator 2402 includes a nonlinearu-shaped distal portion 2418, configured so that distal aperture 2414 isdisposed above side aperture 2416 relative to the bottom of reservoir2404. Accordingly, when upward pressure is applied to fluid channel2412, the upper fluid in reservoir 2404, which is background fluid 2408,is drawn into fluid channel 2412 through distal aperture 2414. At thesame time, the lower fluid in reservoir 2404, which is foreground fluid2406, is drawn into fluid channel 2412 through side aperture 2416. Justas described previously, the intersection of the foreground andbackground fluids in the vicinity of the side aperture results ingeneration of an emulsion of foreground fluid droplets 2418 in thebackground fluid, and the generated emulsion proceeds upward throughchannel 2412 for storage and/or transport.

It should be apparent from the configuration shown in FIG. 64 thatdroplet generator 2402 may be characterized as a “single T” generator,based on the directions of the various incoming and outgoing fluidvelocities. Other configurations, such as a “double T” configuration ora “multi-T” configuration, may be used in conjunction with a dropletgenerator having a u-shaped or similarly shaped tip. By altering thenumber of side apertures, their positions, their sizes, and theirshapes, the resulting emulsion may be given essentially any desiredcharacteristics.

FIG. 65 depicts another droplet generation system, generally indicatedat 2420, which is configured to generate an emulsion of relatively moredense droplets in a background of relatively less dense fluid. System2420 includes a droplet generator 2422 and a fluid reservoir 2424.Droplet generator 2422 is a syringe having a body 2426 that serves as avariable-volume emulsion reservoir, and an elongate sharp tip 2428 thatdefines a fluid channel 2430. The syringe includes a movable plunger2431, which is configured to slide up and down to create pressuredifferences within the syringe and to vary the volume of the emulsionreservoir. They syringe also will include a plunger control mechanism(not shown), such as a handle or plunger head configured to allow a userto move the plunger longitudinally within the body of the syringe.

Droplet generator 2422 includes a distal aperture 2432 at the end of tip2428, configured to receive or expel fluid in fluid channel 2430. Tip2428 also includes a side aperture 2434, also configured to receive orexpel fluid. When negative pressure is exerted (i.e., when a partialvacuum is created) within fluid channel 2430, fluid thus may be drawninto both distal aperture 2432 and side aperture 2434. When fluids ofdifferent densities are disposed in fluid reservoir 2424 (as depicted inFIG. 65), different fluids may be drawn into the two differentapertures, so that tip 2428 acts as a “single T” emulsion generator asdescribed in detail above. Also as described previously, any desirednumber, size and/or shape of side apertures may be used to generate anemulsion having desired properties.

Fluid reservoir 2424 is depicted in FIG. 65 as a substantiallycylindrical chamber having a removable threaded top 2436, which includesa penetrable membrane such as a layered septum 2438. Thus, once adesired amount of emulsion has been produced and drawn into body 2426,droplet generator 2422 may be withdrawn from the fluid reservoir totransport the emulsion to another location such as a thermocyclinginstrument. The fluid reservoir is configured to contain the fluidingredients of a desired emulsion without significant leakage, whileallowing droplet generator 2422 to penetrate the reservoir and establishfluid contact with the fluids in the reservoir. Any alternativereservoir having these features may be used with droplet generator 2422,such as reservoirs of various shapes and sizes, and reservoirs havingvarious alternative types of penetrable membranes.

Droplet generator 2422 is disposed below fluid reservoir 2424 in FIG.65. Accordingly, a relatively high density sample-containing fluid 2440will be disposed in the vicinity of side aperture 2434 of the dropletgenerator, whereas a relatively low-density background fluid 2442 (suchas oil) will be disposed in the vicinity of distal aperture 2432 of thedroplet generator. This results in an emulsion of sample-containingdroplets in an oil background. Of course, system 2420 could be turned180 degrees (i.e., flipped upside down relative to FIG. 65), in whichcase it would be configured to produce an emulsion of sample-containingdroplets in an oil background when the sample-containing fluid is lessdense than the background fluid.

FIG. 66 depicts a lower portion of yet another droplet generationsystem, generally indicated at 2450, which is configured to produce anemulsion of relatively higher density droplets in a background ofrelatively lower density fluid. System 2450 includes a butted tube typedroplet generator 2452, and a fluid reservoir 2454. Fluid reservoir 2454is substantially similar to the fluid reservoirs depicted in FIGS. 61-64and described above, and accordingly will not be described further.Droplet generator 2452 includes a tube 2456 having a distal aperture2458 and a pair of opposing side apertures 2460, 2462. When a partialvacuum is created within tube 2456 from above, higher densitysample-containing fluid 2464 is drawn into distal aperture 2458 andlower density background fluid 2466 is drawn into side apertures 2460,2462. The fluids intersect in the vicinity of the side apertures toproduce droplets 2468 of sample-containing fluid that travel upwardthrough tube 2456 in an emulsion. Due to the directions of fluidvelocity near the side apertures, droplet generator 2452 may becharacterized as a cross-type droplet generator.

F. Example 6

FIG. 67 depicts a lower portion of another cross-type droplet generationsystem, generally indicated at 2480. Droplet generation system 2480includes an emulsion generator 2482, and an emulsion reservoir 2484configured to receive the emulsion generated by the emulsion generator.As its name suggests, emulsion generator 2482 is configured to generatean emulsion of sample-containing droplets, typically in the form ofaqueous droplets in an oil background. Emulsion reservoir 2484 isdepicted in FIG. 67 as a test tube, but more generally may be anyreservoir configured to receive, contain and/or transport an emulsion toa desired location.

Emulsion generator 2482 includes an inner fluid chamber 2486 configuredto contain a sample-containing fluid 2488, and an outer fluid chamber2490 surrounding portions of the inner fluid chamber and configured tocontain a background fluid 2492, typically an oil. The depicted lowerportions of inner fluid chamber 2486 and outer fluid chamber 2490 aresubstantially cylindrical and concentric, but other geometries may bechosen. Inner fluid chamber 2486 includes a distal aperture 2494,configured to allow passage of sample-containing fluid 2488 out of theinner fluid chamber at a desired rate. Outer fluid chamber 2490 includesa distal aperture 2496, configured to allow passage of an emulsion outof the outer fluid chamber at a desired rate. Accordingly, distalapertures 2494, 2496 may have any suitable size and/or shape resultingin desirable flow characteristics through the apertures.

Background fluid channels 2498, 2500 are formed between the lowerexternal boundary of the inner fluid chamber and the lower internalboundary of the outer fluid chamber, and configured to transferbackground fluid 2492 radially inward toward distal aperture 2496 of theouter fluid chamber. In some cases, the lower boundary of inner fluidchamber 2486 may rest directly upon the lower inside surface of outerfluid chamber 2490, except for a pair of grooves forming discrete fluidchannels 2498, 2500. In other cases, inner fluid chamber 2486 and outerfluid chamber 2490 may be held out of direct contact with each other bysome spacing mechanism (not shown). In this case, background fluidchannels 2498, 2500 will be portions of a single circular backgroundfluid channel through which background fluid can move radially inwardtoward aperture 2496.

System 2480 may be operated by applying positive pressure from abovechambers 2486, 2490, to push sample-containing fluid 2488 and backgroundfluid 2492 toward their respective apertures. The inner and outer fluidchambers are positioned so that oil flowing radially inward through thebackground fluid channels will intersect with sample-containing fluidpassing out of the inner fluid chamber through distal aperture 2494 ofthe inner fluid chamber, to generate an emulsion of sample-containingdroplets within the background fluid which will pass through distalaperture 2496 of the outer fluid chamber and into emulsion reservoir2484, where it may be stored or transported as desired. Emulsionreservoir 2484 may at least partially surround the emulsion generator orbe otherwise configured to receive the emulsion generated by theemulsion generator. Typically, emulsion generator 2492 is removable fromemulsion reservoir 2484, and would likely be removed after the emulsionhas been generated. The emulsion generator then may be disposed of orcleaned in preparation for the introduction of a new sample.Alternatively, inner chamber 2486 may be removable from outer chamber2490 and disposable, while outer chamber 2490 may be reusable.

Aside from applying positive pressure to the fluids within chambers 2486and 2490, an emulsion may be formed similarly by applying negativepressure to pull the fluids through apertures 2494 and 2496, forexample, by creating a partial vacuum in the emulsion reservoir. In thecase of either positive or negative pressure, the pressure may becreated through any suitable mechanism such as a pump, a bulb, or aplunger. Furthermore, system 2480 may be placed in a centrifuge andspun, to create an emulsion based on the inertia of the constituentfluids. This technique may sometimes be referred to as causing fluidmotions through “centrifugal force.” When a centrifuge is used in thismanner, system 2480 may be characterized as a “spin column” dropletgenerator or emulsion generator.

FIG. 68 depicts portions of another emulsion generation system,generally indicated at 2520. System 2520 is similar in many respects tosystem 2480 of FIG. 67, and further illustrates the potentiallyremovable and/or disposable nature of various parts of the system.System 2520 includes an emulsion generator 2522 and an emulsionreservoir 2524. Emulsion generator 2522 includes an inner fluid chamber2525 configured to contain a sample-containing fluid 2526, and an outerfluid chamber 2528 configured to contain a background fluid 2530. Innerfluid chamber 2525 and outer fluid chamber 2528 are substantiallycylindrical and concentric. A distal aperture 2532 of the inner fluidchamber is configured to allow passage of sample-containing fluid out ofthe inner fluid chamber, and a distal aperture 2534 of the outer fluidchamber is configured to allow passage of an emulsion out of the outerfluid chamber.

Fluid channels 2536, 2538 are formed between the lower boundary of theinner fluid chamber and the lower inside surface of the outer fluidchamber, and configured to transfer background fluid inward towarddistal aperture 2534. An emulsion 2540 of sample-containing droplets2542 is formed either by applying positive pressure pushsample-containing fluid and background fluid toward their respectiveapertures, or by applying negative pressure to accomplish the samemotions. Pressure may be created by any suitable mechanism such as apump, bulb, plunger, or centrifuge, as described previously with respectto FIG. 67. The generated emulsion passes through aperture 2534 intoemulsion reservoir 2524, for storage or transport to a thermocyclinginstrument.

Emulsion generator 2522 is a self-contained component that may beinserted and removed from emulsion reservoir 2524 as desired. Asupporting lip 2544 of the emulsion generator is configured to overlapside wall 2546 of the emulsion chamber, to support the emulsiongenerator in a desired position with respect to the emulsion chamber.The emulsion generator includes a lid 2548 that may be rotated away fromthe emulsion generator to allow the addition of fluids and/or pressure,and rotated to cover the emulsion generator to form a fluid tight seal.This may allow convenient transport of the emulsion generator, and alsomay allow the use of a centrifuge without undesirable leaking.Similarly, the emulsion reservoir includes a lid 2550 that may be usedto selectively form a fluid tight seal at the top of the emulsionreservoir. This may allow convenient transport, storage or furtherprocessing of an emulsion with substantially no loss of fluid from thereservoir.

G. Example 7

FIG. 69 illustrates the relationship between various cross-type dropletgenerators. More specifically, FIG. 69 shows a first cross-type dropletgenerator 2560 including a single cross, a second cross-type dropletgenerator 2580 including two crosses, a third cross-type dropletgenerator 2600 including three crosses, and a butted tube cross-typedroplet generator 2620.

Droplet generator 2560 includes hollow channels 2562, 2564 thatintersect at an intersection region 2566. To generate droplets, one ofthese channels will generally carry a foreground fluid towardintersection region 2566 from one direction, while the other channelcarries a background fluid toward intersection region 2566 from bothdirections. Typically, channel 2562 will carry a foreground fluid suchas a sample-containing solution, and channel 2564 will carry abackground fluid such as oil, but the opposite is also possible. In anycase, an emulsion will be created at intersection region 2566 and willcontinue moving through channel 2562 in the direction of travel of theforeground fluid, as described in detail above.

Droplet generator 2580 includes three hollow channels 2582, 2584, 2586that intersect at an intersection region 2588. To generate droplets,channel 2582 will typically carry a foreground fluid such as asample-containing solution toward intersection region 2588 from a singledirection, and each of channels 2584, 2586 will typically carry abackground fluid such as oil toward intersection region 2588 from twoopposite directions. In that case, an emulsion will be created atintersection region 2588 and will continue moving through channel 2582in the direction of travel of the foreground fluid. It is also possiblethat each of channels 2584, 2586 would carry a foreground fluid towardintersection region 2588 from a single direction, and channel 2582 wouldcarry a background fluid toward intersection region 2588 from twoopposite directions. In that case, the emulsion created at intersectionregion 2588 would travel through both channels 2584 and 2586, in theoriginal directions of travel of the foreground fluid in each of thosechannels. Droplet generator 2580 thus may function to produce dropletsthat emerge from two separate channels.

Similarly, droplet generator 2600 includes four channels 2602, 2604,2606, 2608 that intersect to generate an emulsion of foreground fluiddroplets in background fluid at an intersection region 2610. By analogyto the three-channel configuration of droplet generator 2580, thefour-channel configuration of droplet generator 2600 may be used eitherto generate a single emulsion that travels through channel 2602, or togenerate multiple emulsions that travel through channels 2604, 2606, and2608.

Droplet generator 2620 is a butted tube generator that includes a firstsection of hollow tube 2622 and a second section of hollow tube 2624.Tube section 2622 includes a fluid channel 2626, and tube section 2624includes a fluid channel 2628. The tube sections are separated by asmall distance, forming an intersection region 2630 between the tubes.Accordingly, if a foreground fluid flows toward intersection region 2630through channel 2626, and a background fluid flows radially inwardtoward intersection region 2630 from the region outside the tubes, anemulsion can be created and flow into channel 2628.

The progression from droplet generator 2560 through droplet generator2620 illustrates the relationship between these various dropletgenerators. Specifically, if the variable n is chosen to represent thenumber of radial fluid channels that intersect a longitudinal fluidchannel at an intersection region within a tube, then droplet generator2560 may be characterized as an “n=1” cross-type droplet generator,droplet generator 2580 may be characterized as an “n=2” cross-typedroplet generator, droplet generator 2600 may be characterized as an“n=3” cross-type droplet generator, and droplet generator 2620 may becharacterized as an “n=0.0” cross-type droplet generator, because thegap between tubes 2622 and 2624 may be viewed as formed from an infinitenumber of radial fluid channels extending continuously around thecircumference of a single elongate tube.

H. Example 8

FIGS. 70 and 71 depict additional cross-type droplet generation systems,which are similar to droplet generation system 2480 of Example 6, butwhich are configured to generate droplets of two or more substantiallydifferent sizes.

FIG. 70 shows a lower portion of a first such cross-type dropletgeneration system, generally indicated at 2640, which is configured togenerate droplets of two substantially different sizes. Accordingly,droplet generation system 2640 includes an emulsion generator 2642, andan emulsion reservoir 2644 configured to receive the emulsion generatedby the emulsion generator. Emulsion reservoir 2644 may be any reservoirconfigured to receive, contain and/or transport an emulsion to a desiredlocation, such as a well, a pipette tip, a spin column or vial, or asyringe body.

Emulsion generator 2642 is configured to generate an emulsion ofsample-containing droplets of two different sizes. Specifically,emulsion generator 2642 includes first and second inner fluid chambers2646, 2648 each configured to contain a sample-containing fluid 2650,and an outer fluid chamber 2652 surrounding portions of the inner fluidchambers and configured to contain a background fluid 2654, such as anoil. Alternatively, inner fluid chambers 2646, 2648 each may contain adifferent fluid, in which case the generated droplets will havedifferent constituents as well as different sizes.

Regardless of their contents, inner fluid chambers 2646, 2648respectively include distal apertures 2656, 2658, configured to allowpassage of sample-containing fluid out of each inner fluid chamber.Outer fluid chamber 2652 includes distal apertures 2660, 2662, eachaligned with one of apertures 2656, 2658. Each pair of aligned aperturesis configured to allow passage of droplets of a particular size, as FIG.70 indicates. Emulsion 2664 created via the aligned apertures of system2640 is otherwise produced in the same way emulsion 2520 is produced indroplet generation system 2480 of FIG. 67, and the details will not berepeated here.

FIG. 71 shows a droplet generation system 2670 much like dropletgeneration system 2640 of FIG. 70, except that system 2670 is configuredto generate droplets across a range of many different sizes.Accordingly, droplet generation system 2670 includes an emulsiongenerator 2672, and an emulsion reservoir 2674 configured to receive theemulsion generated by the emulsion generator. As in many of thepreviously described embodiments, emulsion reservoir 2674 may be anyreservoir configured to receive, contain and/or transport an emulsion toa desired location, such as a well, a pipette tip, a spin column orvial, or a syringe body.

Emulsion generator 2672 is configured to generate an emulsion ofsample-containing droplets of a plurality of different sizes. Emulsiongenerator 2672 thus includes an inner fluid chamber 2676 configured tocontain a sample-containing fluid 2678, and an outer fluid chamber 2679surrounding portions of the inner fluid chamber and configured tocontain a background fluid 2680. Although FIG. 71 depicts only a singleinner chamber 2676, two or more separate inner chambers couldalternatively be used, as in FIG. 70.

Inner fluid chamber 2676 includes a plurality of distal apertures 2682,2684, 2686, 2688, each configured to allow passage of sample-containingfluid out of the inner fluid chamber at a particular rate. Outer fluidchamber 2678 includes distal apertures 2690, 2692, 2694, 2696, eachaligned with one of the apertures of the inner chamber to allow passageof an emulsion including droplets of a particular size. Thus, dropletgeneration system 2670 is configured to generate an emulsion 2698 thatincludes droplets of a wide range of sizes. In a similar manner, adroplet generation system may be configured to produce an emulsionhaving any desired characteristic droplet size distribution.

I. Example 9

This example describes further aspects of exemplary droplet generators.The droplet generation systems described above generally involvemultiple separate components, such as a droplet generator and acomplementary reservoir. However, a droplet generation system accordingto the present disclosure also may take the form of an injection moldedcartridge, with or without sample preparation capabilities. Such acartridge would generally include chambers or protrusions acting as thebarrels of syringes, wells, or reservoirs to contain the sample and oilfor combination into an emulsion of sample-containing droplets. Thesechambers will require sturdy walls that can withstand the side forcesexpected during pumping, insertion of the disposable portion into anon-disposable portion of the system, and shipping/handling. Therefore,the walls of the chambers are envisioned to be approximately 0.020 inchthick but could range in thickness from 0.04 to 0.40 inches.

A disposable cartridge-style droplet generator also would generallyinclude very precise microchannels to contain and direct the flow ofsample-containing solution and oil. These channels could be, forexample, approximately 250 microns wide and 250 microns deep, althoughthese dimensions each could range from approximately 50 microns toapproximately 350 microns. Furthermore, some areas of the dropletgenerator (specifically, those contacting a sample) must bebiocompatible, whereas others areas of the disposable need not meet thisrequirement.

Integrating droplet generation into a single assembly such as adisposable cartridge may have certain efficiency advantages over amulti-component droplet generation system. Specifically, if dropletgeneration involves the use of two or more subassemblies manufacturedseparately, there will typically be more potential for (a) leakage atthe connections between the subassemblies, (b) increased unswept volumesin those connections, (c) more volume in the lines connection, (d)greater complexity in the fluid circuit, and (e) increasefabrication/assembly costs. On the other hand, integrating these diverserequirements into a single assembly results in potential savings in allthe areas listed.

A molded droplet generator cartridge also may have various otheradvantageous features. For example, moldable plastic typically hasminimal or no absorption of material such as protein, DNA, RNA, lipids,or other constituents of biological samples expected to be tested.Furthermore, it is possible to mold protrusions able to withstand sideforces on one side of a part and microfluidic channels on the oppositeside, as part of a single molding step. A plate, thin sheet, or foil ofthe same or similar material is then bonded to the side of the part withmicrofluidic channels, resulting in tube-like channels connectingvarious areas of the assembly. Holes through the part connect the barreltype features to the channels. This means that all alignments betweenthese features can be inexpensively manufactured, since they are moldedinto one structure.

The anticipated average operating pressures within a disposable dropletgenerating cartridge are 2 to 5 psi. By keeping the fluid pressuresrelatively low, a single molded cartridge can meet the diverse functionslisted elsewhere in this disclosure. Maintaining lower internaloperating pressures rather than higher pressures also means that thecartridge can have (a) thinner wall sections (i.e., less need for strongstructures to withstand breakage), (b) less bulging of the walls (i.e.,more uniformity in controlling fluid flows with pressure variations),and (c) thinner plates bonded to the microchannel side of the cartridge.These factors result in decreased production assembly times and deceasedproduct cost.

Depending on whether a disposable cartridge-type droplet generator isused to generate water-in-oil emulsions or multiple emulsions, it may bedesirable for the fluid contacting surfaces of the droplet generator tobe either hydrophobic or hydrophilic. Either of these alternatives maybe accomplished by choosing an appropriate material that is compatiblewith the molding process, and/or by applying a coating to alter surfaceproperties of the chosen material.

J. Example 10

This example describes additional aspects of droplet generation, inaccordance with aspects of the present disclosure, presented withoutlimitation as a series of numbered sentences.

1. A droplet generator system, comprising (A) a droplet outlet portionincluding an emulsion outlet channel and upper and lower channel wallsdefining an oil channel; and (B) a sample-containing portion configuredto be selectively assembled with the droplet outlet portion andincluding (i) a sample reservoir; and (ii) a fluid outlet apertureconfigured to emit droplets of sample-containing fluid from the samplereservoir; wherein when the droplet outlet portion and thesample-containing portion are assembled together, wherein asubstantially fluid tight seal is formed between the droplet outletportion and the sample-containing portion; and wherein droplets emittedby the fluid outlet aperture intersect oil traveling in the oil channelto produce an emulsion of water-in-oil droplets that passes into theemulsion outlet channel.

2. The system of claim 1, wherein the sample-containing portion isconfigured to be a single-use, disposable component of the system.

3. The system of claim 2, wherein the sample-containing portion isconstructed of injection-molded thermoplastic.

4. A droplet generation system, comprising (A) a fluid reservoirconfigured to hold a background emulsion fluid having a first densityand a foreground emulsion fluid having a second density; and (B) adroplet generator including an elongate tip configured to be at leastpartially inserted into the fluid reservoir and having at least one sideaperture and a distal aperture; wherein the distal aperture isconfigured to be in contact with background fluid held by the reservoirand the side aperture is configured to be in contact with foregroundfluid held by the reservoir when the reservoir contains background andforeground fluids and the elongate tip is inserted into the reservoir;and wherein the droplet generator is configured so that foreground fluidflowing into the side aperture intersects with a stream of backgroundfluid that enters the tip through the distal aperture, to form anemulsion of foreground fluid droplets in background fluid.

5. The droplet generation system of paragraph 4, wherein the dropletgenerator further includes an emulsion chamber configured to receive theemulsion.

6. The droplet generation system of paragraph 4, wherein the at leastone side aperture includes a plurality of side apertures.

7. The droplet generation system of paragraph 4, wherein the elongatetip includes a u-shaped distal portion.

8. A droplet generation system, comprising (A) an emulsion generatorincluding (i) an inner fluid chamber configured to contain asample-containing fluid and having a distal aperture configured to allowpassage of the sample-containing fluid out of the inner fluid chamber;and (ii) an outer fluid chamber configured to contain a backgroundfluid, the outer fluid chamber surrounding at least portions of theinner fluid chamber and having a distal aperture configured to allowpassage of an emulsion out of the outer fluid chamber; whereinbackground fluid channels are formed between an external boundary of theinner fluid chamber and an internal boundary of the outer fluid chamber,and configured to transfer background fluid radially inward toward thedistal aperture of the outer fluid chamber; and wherein the inner andouter fluid chambers are positioned so that oil flowing radially inwardthrough the background fluid channels will intersect withsample-containing fluid passing out of the inner fluid chamber throughthe distal aperture of the inner fluid chamber, to generate an emulsionof sample-containing droplets within the background fluid which willpass through the distal aperture of the outer fluid chamber; and (B) anemulsion reservoir at least partially surrounding the emulsion generatorand configured to receive the emulsion generated by the emulsiongenerator.

V. CONTINUOUS FLOW THERMOCYCLER

This Section describes exemplary thermocyclers, for example, for use indroplet-based assays.

It may be desirable, in systems such as DNA amplification systems, toperform temperature-dependent reactions for increasing the number ofcopies of a sample, or component(s) thereof. Methods of cyclicallyvarying the temperature of a fluid or other material generally may betermed methods of “thermocycling,” and an apparatus used to accomplishsuch cyclical temperature variations generally may be termed a“thermocycler.” In the case of DNA amplification through PCR, cyclicaltemperature changes cause repeated denaturation (also sometimes termedDNA “melting”), primer annealing, and polymerase extension of the DNAundergoing amplification. Typically, twenty or more cycles are performedto obtain detectable amplification. In other processes, such asalternative enzymatic amplification processes, thermocycling may haveother effects, and different temperature ranges and/or different numbersof temperature changes may be appropriate.

FIG. 72 is a flowchart depicting a method, generally indicated at 3100,of thermocycling a sample/reagent emulsion or other fluid mixture topromote PCR. Typically, three separate temperatures or temperatureranges are provided to the fluid to accomplish thermocycling for PCR.Other numbers of temperature ranges, such as one, two, four, or more,may be provided for different processes. In the case of PCR, providing afirst, relatively higher temperature to the fluid, as indicated at step3102, causes the target DNA to become denatured. This denaturingtemperature is typically in the range of 92-98° C. Providing a second,relatively lower temperature to the fluid, as indicated at step 3104,allows annealing of DNA primers to the single-stranded DNA templatesthat result from denaturing the original double-stranded DNA. Thisprimer annealing temperature is typically in the range of 50-65° C.Finally, providing a third, middle temperature to the fluid, asindicated at step 3106, allows a DNA polymerase to synthesize a new,complementary DNA strand starting from the annealed primer. Thispolymerase extension temperature is typically in the range of 70-80° C.,to achieve optimum polymerase activity, and depends on the type of DNApolymerase used.

In some cases, a single temperature may be provided for both primerannealing and polymerase extension (i.e., steps 3104 and 3106 above),although providing a single temperature for these processes may notoptimize the activity of the primers and/or the polymerase, and thus maynot optimize the speed of the PCR reaction. When provided for bothannealing and extension, this single temperature is typically in therange of 55-75° C.

A PCR thermocycler also may include, in addition to the two or threetemperature zones described above, an integrated or complementary “hotstart” mechanism configured to provide a relatively high hot-starttemperature, as indicated at step 3108. The hot-start temperature isprovided to initiate PCR and/or to prepare a sample/reagent mixture forinitiation of PCR upon the addition of a suitable polymerase. Morespecifically, providing a hot-start temperature may reverse theinhibition of a polymerase enzyme that has been added to inhibit primingevents that might otherwise occur at room temperature. In this case,heating the sample/reagent mixture to a hot-start temperature initiatesthe onset of PCR. In other instances, providing a hot-start temperaturemay preheat the sample and the primers in the absence of the polymerase,in which case subsequent addition of the polymerase will initiate PCR.The hot start temperature is typically in the range of 95-98° C.

The thermocycler also may include integrated or complementary mechanismsfor allowing “final elongation” and/or “final hold” steps, afterthermocycling has (nominally) been completed. For example, in the formercase, the thermocycler may include a mechanism configured to maintainsamples at the extension temperature long enough (e.g., for 5-15minutes) to ensure that any remaining single-stranded nucleotide isfully extended. In continuous flow systems, this mechanism may include arelatively long piece of narrow tubing to increase path length, and/or arelatively short piece of wider tubing to decrease flow rate, bothmaintained at an extension temperature. Alternatively, or in addition,the thermocycler may include a mechanism for holding or storing samples(e.g., for an indefinite time) at a temperature below the extensiontemperature (e.g., 4-15° C.).

Various methods of providing the desired temperatures or temperatureranges to a sample/reagent fluid mixture may be suitable for PCR. Forexample, a fluid may be disposed within one or more stationary fluidsites, such as test tubes, microplate wells, PCR plate wells, or thelike, which can be subjected to various temperatures provided in acyclical manner by an oven or some other suitable heater acting on theentire thermal chamber. However, such array-type PCR systems may belimited by the number of fluid sites that can practically be fluidicallyconnected to the system and/or by the kinetics of changing temperaturesin a large (high-thermal-mass) system (e.g., transition times betweenmelt, anneal, and extension temperatures in commercial systems may beorders of magnitude longer than the fundamental limits of Taq polymeraseprocessivity). Alternatively, fluid may be passed continuously orquasi-continuously through various temperature regions, in a cyclicalmanner. In this case, it is desirable to minimize heat transfer betweenthe regions, to provide sharp temperature transitions between theregions. It is also desirable to monitor the temperature of each regioncontinuously and to provide rapid feedback to maintain a relativelyconstant desired temperature in each region.

One type of continuous-flow PCR system involves coiling or windingfluidic tubing to form a fluid channel in a helical shape around athermocycler that is configured to provide the various desiredtemperatures or temperature regions. Furthermore, various alternativesto externally wrapped fluidic tubing may be used to provide a fluidchannel configured to transport an emulsion of sample-containingdroplets cyclically through various temperature regions. For example,tubing may be disposed within the body of thermocycler, such as bycasting the thermocycler (or the inner segments of the thermocycler)around the tubing. Alternatively, a fluid tight coating (such as asilicon coating) may be applied to external grooves or channels of thethermocycler and then wrapped with a fluid tight sheet (such as asilicon sheet), to define an integrated fluid channel passing cyclicallyaround the thermocycler without the need for any separate tubing at all.

Thus, providing the first, second, third and/or hot-start temperaturesat steps 3102, 3104, 3106, 3108 of method 3100 may include transportingan emulsion in a substantially helical path cyclically through adenaturing temperature region, a primer annealing temperature region, apolymerase extension temperature region, and/or a hot-start temperatureregion of the thermocycler. These various temperature regions may bethermally insulated from each other in various ways, and each region mayprovide a desired temperature through the use of resistive heatingelements, thermoelectric coolers (TECs) configured to transfer heatbetween a thermal core and the temperature regions, and/or by any othersuitable mechanism. Various heat sinks and sources may be used toprovide and/or remove heat from the thermocycler, either globally (i.e.,in substantial thermal contact with two or more temperature regions) orlocally (i.e., in substantial thermal contact with only one temperatureregion).

The following examples describe specific exemplary methods and apparatusfor cyclically heating and cooling a sample/reagent mixture tofacilitate DNA amplification through PCR, i.e., exemplary thermocyclersand methods of thermocycling suitable for PCR applications. Additionalpertinent disclosure may be found in the U.S. provisional patentapplications listed above under Cross-References and incorporated hereinby reference, particularly Ser. No. 61/277,200, filed Sep. 21, 2009.

A. Selected Embodiments 1

This Section describes a first exemplary thermocycler 3200, inaccordance with aspects of the present disclosure; see FIGS. 73-80.

FIG. 73 is an exploded isometric view of key components of thermocycler3200. The thermocycler includes a core 3202 defining a centrallongitudinal axis, three inner segments 3204, 3206, 3208, and threeouter segments 3210, 3212, 3214. The three pairs of segments correspondto the three portions of the PCR thermal cycle described above, inconnection with FIG. 72, and define the corresponding temperatureregions. Specifically, segments 3204 and 3210 correspond to the meltphase, segments 3206 and 3212 correspond to the anneal phase, andsegments 3208 and 3214 correspond to the extension (extend) phase,respectively. In alternative embodiments, the thermocycler could includealternative numbers of segments, for example, two segments in athermocycler in which the annealing and extension phases were combined.Collectively, portions or regions of the thermocycler involved inmaintaining particular temperatures (or temperature ranges) may betermed “temperature regions” or “temperature-controlled zones,” amongother descriptions.

FIG. 74 is an unexploded isometric view of a central portion of thethermocycler of FIG. 73, emphasizing the relationship between the coreand inner segments. Core 3202 is configured as both a heat source and aheat sink, which can be maintained at a constant desired temperatureregardless of whether it is called upon to supply or absorb heat. Forexample, in some embodiments, core 3202 may be maintained atapproximately 70° Celsius. However, more generally, in embodiments inwhich the core acts as a heat source and a heat sink between two or moresegments, the core may be maintained at any suitable temperature betweenthe temperatures of the warmest and coolest segments (e.g., between thetemperature of the melt segment and the annealing segment).

Inner segments 3204, 3206, 3208 are attached to the core and configuredto form an approximate cylinder when all of the inner segments areattached or assembled to the core. Inner segments 3204, 3206, 3208 areequipped with external grooves 3216 on their outer peripheral surfaces,as visible in FIGS. 73 and 74. When the inner segments are assembled tothe core, these grooves form a helical pattern around the circumferenceof the cylindrical surface formed by the inner segments. Grooves 3216are configured to receive fluidic tubing that can be wrappedcontinuously around the inner segments, as described below, to allow afluid traveling within the tubing to travel helically around thecircumference formed by the assembled inner segments. The fluidic tubingacts as a fluid channel to transport an emulsion of sample-containingdroplets cyclically through the various temperature regions of thethermocycling system.

Outer segments 3210, 3212, 3214 are configured to fit closely around theinner segments, as seen in FIG. 73. Thus, the fluidic tubing may bewound between the inner and outer segments and held in a stable, fixed,environmentally controlled position by the segments.

FIG. 75 is an isometric magnified view of a portion of the assembledthermocycler. This embodiment is particularly suitable for relativelysmall outer diameter fluidic tubing. Portions of outer segments 3210,3214 are disposed around inner segments 3204, 3208 and core 3202 (notvisible). Fluidic tubing 3218 can be seen disposed in grooves 3216,which are partially visible within an aperture 3220 formed by the outersegments. Additional fastening apertures 3222 are provided in the outersegments to facilitate attachment of the outer segments to the innersegments. The tubing may pass from outside to inside thermocycler 3200through an ingress region 3224. The tubing is then wrapped helicallyaround the inner segments a minimum number of times, such as 20 or moretimes, after which the tubing may pass from inside to outsidethermocycler 3200 through an egress region 3226. Egress region 3226 isrelatively wide, to allow the tubing to exit thermocycler 3200 afterforming any desired number of coils around the inner segments.

FIG. 76 is an isometric magnified view of a portion of an alternativeembodiment of the assembled thermocycler. This embodiment, which shows aslight variation in the shape of the outer segments, is particularlysuitable for relatively large outer diameter fluidic tubing.Specifically, FIG. 76 shows outer segments 3210′, 3214′ disposed aroundinner segments 3204′, 3208′ and core 3202. Grooves 3216′, which arerelatively wider than grooves 3216 of FIG. 75, are partially visiblewithin an aperture 3220′ formed by the outer segments. In FIG. 76,fluidic tubing may pass from outside to inside thermocycler 3200 andvice versa at any desired groove positions, simply by overlapping theedge of aperture 3220′ with the tubing. Between the ingress and egresstubing positions, the tubing may be wrapped around the inner segments tomake any desired number of helical coils around the inner segments.

FIG. 77 is a top plan view of the assembled thermocycler, without theouter segments attached. This view shows three thermoelectric coolers(TECs) 3228, 3230, 3232 disposed between core 3202 and inner segments3204, 3206, 3208. One of these, TEC 3228, can be seen in FIG. 73. EachTEC is configured to act as a heat pump, to maintain a desiredtemperature at its outer surface when a voltage is applied across theTEC. The TECs may be set to steady-state temperatures using a suitablecontroller, such as a proportional-integral-derivative (PID) controller,among others. The TECs operate according to well-known thermoelectricprinciples (in which, for example, current flow is coupled with heattransfer), such as the Peltier effect, the Seebeck effect, and/or theThomson effect. The TECs may be configured to transfer heat in eitherdirection (i.e., to or from a specific thermocycler element), with oragainst a temperature gradient, for example, by reversing current flowthrough the TEC. Thus, the TECs may be used to speed up or enhanceheating of an element intended to be warm, speed up or enhance coolingof an element intended to be cool, and so on, to maintain eachtemperature region approximately at a different desired temperature.Suitable TECs include TECs available from RMT Ltd. of Moscow, Russia.

Each TEC, in turn, may be sandwiched between a pair of thermallyconductive and mechanically compliant pads 3234, as seen in FIGS. 73 and77. Pads 3234 may be configured to protect the TECs from damage due tosurface irregularities on the outer surface of core 3202 and in theinner surfaces of inner segments 3204, 3206, 3208. Alternatively, or inaddition, pads 3234 may be configured to minimize the possibility ofpotentially detrimental shear stresses on the TECs. Suitable padsinclude fiberglass-reinforced gap pads available from the BergquistCompany of Chanhassen, Minn.

FIG. 78 is a schematic section diagram depicting the relativedisposition of core 3202, TECs 3228, 3230, 3232, inner segments 3208,3206, 3204, and tubing 3218. Here, the core, TECs, and inner segmentsare collectively configured to maintain the outer surfaces 3236, 3238,3240, respectively, of the inner segments at any desired temperatures tofacilitate PCR reactions in fluids passing through tubing disposedhelically around the cylindrical perimeter of the assembled innersegments. FIG. 78 can be thought of as the top view shown in FIG. 77,cut along line C in FIG. 77 and shown “unrolled” into a representativelinear configuration. FIG. 78 can be obtained from FIG. 77 by continuousdeformation, making these figures topologically equivalent(homeomorphic), and meaning that FIG. 78 may simply be viewed as analternate way of visualizing the arrangement of components shown in FIG.77.

TECs 3228, 3230, and 3232 are configured to maintain outer surfaces3236, 3238, 3240, respectively, of the inner segments at varioustemperatures corresponding to the different stages of PCR, as depictedin FIG. 78. Because tubing 3218 is in thermal contact with outersurfaces 3236, 3238, 3240, the temperature of any fluid in tubing 3218also may be controlled via the TECs. Specifically, outer surface 3236 ismaintained at a temperature Tmeit suitable for melting (or denaturing)DNA, outer surface 3238 is maintained at a temperature Tanneai suitablefor annealing primers to single-stranded DNA templates, and outersurface 3240 is maintained at a temperature Textend suitable forsynthesizing new complementary DNA strands using a DNA polymerase.

TECs 3228, 3230, 3232 respond relatively rapidly to electrical signalsand are independently controllable, so that the desired temperatures atouter surfaces 3236, 3238, 3240 may be maintained relatively accurately.This may be facilitated by temperature sensors that monitor thetemperatures of the outer surfaces and provide real-time feedbacksignals to the TECs. Maintaining the various temperatures is alsofacilitated by gaps 3242, 3244, 3246, which are visible in both FIG. 77and FIG. 78, between the inner segments. These gaps, which in thisexample are filled simply with air, provide insulation between theneighboring inner segments to help keep the inner segments thermallywell-isolated from each other. In other embodiments, the gaps may befilled with other materials.

FIG. 79 is a magnified isometric view of a central portion of grooves3216 and tubing 3218 of FIG. 75, spanning the interface between two ofthe inner segments of the thermocycler. The features of the groovesshown in FIG. 79 are also present in grooves 3216′ of FIG. 76.Specifically, grooves 3216 and 3216′ include sloping edge contours 3248disposed at the periphery of each inner segment 3204, 3206, 3208. Edgecontours 3248 allow the tubing to be wrapped around the inner segments,even if there is a slight misalignment of two of the inner segments withrespect to each other, because the edge contours do not include sharpedges that can be fracture points for tubing under stress from curvaturedue to potential misalignment.

The configuration of the inner segments in this example provides thateach inner segment 3204, 3206, 3208 is substantially thermally decoupledfrom the other inner segments, as FIG. 78 illustrates schematically.This has advantages over systems in which the various temperatureregions are in greater thermal contact, because in this exemplaryconfiguration there is relatively little heat conduction betweensegments. One source of conduction that still exists is conduction viathe fluid and fluidic tubing that passes from one inner segment to thenext; however, as described below, the effects of this conduction ontemperature uniformity are generally small.

FIG. 80 shows actual measured temperature versus arc length, as afunction of average fluid velocity, near the interface between two innersegments configured according to this example. In particular, theeffects of fluid heat conduction on temperature uniformity generallybecome insignificantly small within a few one-thousandths of a radianfrom the interface between inner segments, even for relatively rapidfluid velocity. Cycle times in the system can be adjusted dynamically bychanging the flow rate through either software or hardware modifications(e.g., pump settings, drum radius, arc length of each segment (sincelength of time in a given segment or zone is proportional to the arclength of that segment), capillary internal diameter, etc.).

FIGS. 73 and 77 each show aspects of a mounting system for TECs 3228,3230, 3232. Here, one TEC is mounted between core 3202 and each of innersegments 3204, 3206, 3208, as described previously. To attain positionalaccuracy when attaching each inner segment to the core, locating pins3250 are configured to attach to both the core and one of the innersegments, to align each segment precisely with the core. Furthermore,the presence of the locating pins should reduce the likelihood thatshear forces will act on the TECs and potentially damage them. Thelocating pins fit into complementary pin apertures 3252 disposed in boththe inner segments and the core. In the exemplary embodiment of FIG. 73,a single locating pin is positioned at one end of the core (the top endin FIG. 73), and two locating pins are positioned at the other end ofthe core (the bottom end in FIG. 73).

FIG. 73 also shows bolts 3254 and washers 3256 configured to attach theinner segments to the core. The bolts are generally chosen to have lowthermal conductivity, so that the TECs remain the only significant heatconduction path between the core and the inner segments. For instance,the bolts may be constructed from a heat-resistant plastic or arelatively low thermal conductivity metal to avoid undesirable thermalconduction. The washers may be load compensation washers, such asBelville-type washers, which are configured to provide a knowncompressive force that clamps each inner segment to the core. Thisbolt/washer combination resists loosening over time and also allowsapplication of a known stress to both the bolts and the TECs, leading togreater longevity of the thermocycler.

B. Selected Embodiments 2

Various modifications and/or additions may be made to the exemplaryembodiments of FIGS. 73-80 according to the present disclosure. Forexample, a “hot start” mechanism may be added to facilitate ahigh-temperature PCR activation step. FIG. 81 shows a central portion(i.e., outer segments not shown) of an exemplary thermocycler 3200′including a hot start region 3258, which is separated from the remainderof the thermocycler by a gap 3260. The hot start region is configured toaccept fluidic tubing just as are the inner segments, but is separatedfrom the inner segments by gap 3260 to avoid unwanted heat conductionbetween the hot start region and the other portions of the thermocycler.A separate core portion (not shown) may be configured to heat region3258 to a relatively high activation temperature, typically in the rangeof 95-98° C., to dissociate any polymerase inhibitors that have beenused to reduce non-specific or premature PCR amplification.

Aside from hot start region 3258 and its associated gap and coreportion, the remainder of thermocycler 3200′, which is generallyindicated at 3262, may have a similar construction to thermocycler 3200described previously. Alternatively, instead of thermoelectriccontrollers, thermocycler 3200′ may include an air core surrounded by aplurality of resistive section heaters (not shown) for heating varioustemperature regions 3263, 3265, 3267 of the thermocycler. These regionsmay be separated by insulating gaps 3269, 3271, which extend into aninsulating base portion 3273 to help thermally isolate the temperatureregions from each other. The configuration of the base portion,including the insulating gaps, can be changed to adjust thermalconductance between the different temperature regions.

C. Selected Embodiments 3

This subsection describes various alternative exemplary thermocyclers3202 a-h, in accordance with aspects of the present disclosure; seeFIGS. 82-89.

FIGS. 82-89 are schematic diagrams depicting top views of thethermocyclers. These diagrams, like FIG. 78, correspond to and aretopologically equivalent to three-dimensional cylindrical thermocyclingunits. The thermocyclers each include three inner (e.g., melt, anneal,and extend) segments 3204 a-h, 3206 a-h, 3208 a-h in thermal contactwith fluidic tubing 3218 a-h for carrying samples undergoing PCR. Thesegments, in turn, each may (or optionally may not) be in thermalcontact with respective (e.g., melt, anneal, and extend) heatingelements 3252 a-h, 3254 a-h, 3256 a-h (denoted by vertical bars) fordelivering heat to the segments. The segments also may be in direct orindirect contact with one or more TECs (indicated by cross-hatching),one or more thermal conductive layer(s) (indicated by stippling), one ormore thermal insulating layer(s) (indicated by dashed-dotted hatching),and/or one or more heated or unheated cores (indicated by hatching orstippling, respectively). These and other components of thethermocyclers may be selected and initially and/or dynamically adjustedto establish, maintain, and/or change the absolute and relativetemperatures of the different segments and thus of the associatedfluidic tubing and PCR samples. Specifically, the components may beselected and/or adjusted to accomplish a temperature goal by accountingfor heat added to or removed from the segments via conduction throughother core components (including fluidic tubing and the associatedfluid) and/or convection with the environment. In particular, the TECs,where present, may transfer heat to or from the segments to facilitatemore rapid and precise control over the associated segment temperaturesand thus the associated reaction temperatures.

FIG. 82 depicts a first alternative thermocycler 3200 a. In thisembodiment, the melt, anneal, and extend segments 3204 a, 3206 a, and3208 a are in thermal contact with a common unheated (e.g., plasticblock) core 3260 via respective thermal insulating layers 3264, 3266,3268. The insulating layers (and insulating layers described elsewherein this Section) independently may be made of the same or differentmaterials, with the same or different dimensions, such that the layersmay have the same or different thermal conductivities. For example, inthis embodiment, the insulating layers for the melt and extend segmentsare made of the same material, with the same thickness, whereas theinsulating layer for the anneal segment is made of a different material,with a different thickness. Heat for performing PCR is supplied to thesegments by heating elements 3254 a, 3256 a, 3258 a. This embodiment isparticularly simple to construct, with relatively few, mostly passivecomponents. However, it is not as flexible or responsive as the otherpictured embodiments.

FIG. 83 depicts a second alternative thermocycler 3200 b. In thisembodiment, the melt, anneal, and extend segments 3204 b, 3206 b, and3208 b are in thermal contact with a common heated (e.g., copper) core3270. However, disposed between the segments and the core, preventingtheir direct contact, are respective insulating layers 3274, 3276, 3278(one for each segment), a common thermal conductor 3280 (in contact withall three insulating layers), and a common TEC 3282 (in contact with thecommon thermal conductor and with the common heated core). Heat forperforming PCR is supplied to the segments by heating elements 3254 b,3256 b, 3258 b and by the common core. The TEC may be used to transferheat to and from the inner segments and the heated core, across theintervening insulating and conducting layers, to adjust, up or down, thetemperatures of the segments.

FIG. 84 depicts a third alternative thermocycler 3200 c. In thisembodiment, the melt and extend segments 3204 c and 3208 c are inthermal contact with a common unheated core 3290 via respectiveinsulating layers 3294, 3298, whereas the anneal segment 3206 c is inthermal contact with a heated core 3300 via a dedicated intervening TEC3296. This configuration substantially thermally decouples the annealsegment from the melt and extend segments and allows the temperature ofthe anneal segment to be changed relatively rapidly via heating element3256 c, heated core 3300, and the TEC. The temperatures of the melt andextend segments, which are thermally connected through unheated core3284, may be changed via heating elements 3254 c, 3258 c (to add heat)and conduction to the unheated core (to remove heat).

FIG. 85 depicts a fourth alternative thermocycler 3200 d. In thisembodiment, thermocycler 3200 c (from FIG. 84) is further coupled to acommon heated core 3302 via an intervening TEC 3304, allowing enhancedfeedback and control over the temperatures of the melt and extendsegments via the TEC layer.

FIG. 86 depicts a fifth alternative thermocycler 3200 e. In thisembodiment, the melt, anneal, and extend segments 3204 e, 3206 e, 3208 eare in thermal contact with a common heated core 3310 via either adedicated insulating layer 3314, 3318 (in the case of the melt andextend segments) or a dedicated TEC layer 3316 (in the case of theanneal layer). This configuration allows relatively rapid feedback andcontrol over the temperature of the anneal segment via a combination ofthe heating element 3256 e and the TEC, while still providing a measureof control over the temperatures of the melt and extend segments viaheating elements 3254 e, 3258 e.

FIG. 87 depicts a sixth alternative thermocycler 3200 f. In thisembodiment, which is similar to thermocycler 3200 e of FIG. 86, a commonconducting layer 3320 and a common TEC 3322 separate the segments fromthe entirety of a heated thermal core 3323. The TEC is in thermalcontact with the anneal segment through the conducting layer, whereasthe TEC is separated from the melt and extend segments both by theconducting layer and by dedicated insulating layers 3324, 3328.

FIG. 88 depicts a seventh alternative thermocycler 3200 g. In thisembodiment, the melt, anneal, and extend segments 3204 g, 3206 g, 3208 geach are in thermal contact with a respective heated core 3334, 3336,3338 via a dedicated intervening TEC 3344, 3346, 3348 (for a total ofthree segments, three heated cores, and three TECs). This embodimentprovides rapid feedback and separate control over the temperature ofeach inner segment. In particular, each segment is independently inthermal contact with dedicated heating element and a dedicated heatedcore, such that heat can be transferred to or from the segment from twodedicated sources or sinks. However, this embodiment also is morecomplicated, requiring controllers for each TEC.

FIG. 89 depicts an eighth alternative thermocycler 3200 h. In thisembodiment, in which a single section of a heated core 3354 is alignedinterior to one inner segment (e.g., the extend segment 3208 h) of thethermocycler, separated from the segment by a TEC 3358. The extendsegment, in turn, is in thermal contact with a neighboring inner segment(e.g., the anneal segment 3206 h) via an unheated conductor 3362, whichis separated from the inner segment by a second TEC 3364. The annealsegment, in turn, is in thermal contact with a neighboring inner segment(e.g., melt segment 3204 h) via another unheated conductor 3368, whichis separated from the inner segment by a third TEC 3370. Thus, coresection 3354 remains available to all of the TECs as a heat source andheat sink.

D. Selected Embodiments 4

This example describes a thermocycler disposed within an instrument thatalso includes other components such as a cooling mechanism and aprotective housing; see FIG. 90.

FIG. 90 generally depicts an exemplary thermocycling instrument 3400 atvarious stages of assembly. Instrument 3400 includes a thermocycler,generally indicated at 3402, which is substantially similar tothermocycler 3200 described above, but which generally may take variousforms, including one or more features of any of the thermocyclersdescribed in the previous examples. The instrument may includeadditional components, such as a front plate, connection port, a heatsink, a cooling fan, and/or a housing, as described below.

A front plate 3404 is attached to the thermocycler with a plurality offasteners 3406 that pass through central apertures 3408 in the frontplate and complementary apertures in the thermocycler. The front platehelps to isolate the thermocycler from external air currents and thus tomaintain controlled temperature zones within the unit.

A connection port 3412 is attached to the front plate, and is configuredto supply power to the instrument and to receive sensor informationobtained by the instrument. Thus, the connection port is configured toreceive electrical power from outside the instrument and transmit thepower to the instrument, and to receive sensor signals from within theinstrument and transmit the signals outside the instrument. Transfer ofpower and sensor signals may be accomplished through suitable connectingwires or cables (not shown) disposed within and outside the instrument.

A heat sink 3414 and a cooling fan 3416, which will be collectivelyreferred to as a cooling mechanism 3418, are shown attached to a side ofthe thermocycler opposite the front plate. One or both components ofcooling mechanism 3418 will generally be mounted to the thermocyclerusing suitable fasteners such as bolts, pins and/or screws. In FIG. 90,heat sink 3414 is attached directly to the thermocycler, and cooling fan3416 is attached to the heat sink. Heat sink 3414 includes a centralaperture 3420, which is aligned with a central aperture of thethermocycler core that defines a central longitudinal axis (see, e.g.,FIGS. 73, 74, and 77). These aligned apertures facilitate heat transferfrom the central (axial) portion of thermocycler 3402 into the heatsink. The heat sink also may be formed of a relatively thermallyconductive material to facilitate conduction of excess heat away fromthe thermocycler, and includes convection fins 3424 to facilitateconvection of heat away from the thermocycler.

Cooling fan 3416 is configured to blow cooling air through fins 3424 andaperture 3420 of the heat sink, to increase convective heat transferaway from the heat sink. Air from fan 3416 also may flow or be directedthrough the heat sink and into the central aperture of thermocycler3402, to provide a convection current within the thermocycler. Dedicatedstructures such as baffles, angled walls or canted fins (not shown) maybe provided to facilitate the transfer of air from the cooling fan intothe thermocycler.

Thermocycler 3402 and cooling mechanism 3418 are mounted within anexternal housing, generally indicated at 3426. Housing 3426 may includeseveral discrete sections 3428, 3430, 3432, 3434, which are configuredto conform to various portions of the thermocycler and the coolingmechanism, and which are further configured to fit together andinterface with front plate 3404 to form housing 3426. The variousdiscrete sections and the front plate of housing 3426 are collectivelyconfigured to insulate the thermocycler from external air currents andother factors that could lead to uncontrolled temperature variationswithin the thermocycler.

E. Selected Embodiments 5

This example describes exemplary thermocyclers having temperatureregions that vary in size and/or number along the length of thethermocycler, in accordance with aspects of the present disclosure; seeFIGS. 91-92.

FIG. 91 shows a side elevational view of portions of an exemplarythermocycler, generally indicated at 3450, having three connectedsegments 3452, 3454, 3456, each defining a different temperature region.Segments 3452, 3454, 3456 may be connected via a common core or throughmaterials (typically thermally insulating materials), not shown,disposed between the segments. Segments 3452, 3454, 3456 are angledalong the length of the thermocycler (i.e., along the longitudinalaxis), so that the inner segments of thermocycler 3450 collectively forma generally frustoconical shape as FIG. 91 depicts. Accordingly, eachwinding of fluidic tubing 3458 wrapped around the exterior ofthermocycler 3450 will be progressively shorter from top to bottom inFIG. 91, so that the helical path followed by the tubing decreases inlength over successive cycles. Assuming fluid flows through tubing 3458at a uniform speed, fluid within the tubing will therefore spendprogressively less time in the temperature regions defined by segments3452 and 3456. On the other hand, segment 3454 has a substantiallyconstant width, so that fluid flowing through tubing 3458 will spend asubstantially constant amount of time in the corresponding temperatureregion with each successive cycle, again assuming the fluid flows with auniform speed.

The thermocycler depicted in FIG. 91 may be useful, for example, when itis desirable to begin a thermocycling operation with relatively longtime duration cycles, and subsequently to decrease the cycle duration tospeed up the overall thermocycling process. In applications such as PCR,this may be the case because efficient target molecule replicationbecomes increasingly less important with each successive thermocycle.For instance, if a single target molecule fails to replicate during thefirst cycle and then replicates with perfect efficiency in thesubsequent 19 cycles, the result after 20 cycles will be 2¹⁹ targetmolecules. However, if a single target molecule replicates with perfectefficiency for the first 19 cycles, but one molecule fails to replicateduring the twentieth cycle, the result after 20 cycles will be (2²⁰−1)target molecules.

Aside from a frustoconical shape, many other thermocycler configurationscan be used to affect the time of passage of a sample fluid through thevarious temperature regions of a thermocycler. For example, the sizes ofvarious temperature regions may be decreased in discrete steps, bysequentially decreasing the radius of a cylindrical thermocycler indiscrete steps. In general, any configuration that results in a changingpath length traveled by successive windings of fluidic tubing may besuitable for altering the time a fluid spends at each desiredtemperature over the course of the entire thermocycling process.

FIG. 92 shows a side elevational view of portions of an exemplarythermocycler, generally indicated at 3500, having temperature regionsthat vary in number along the length of the thermocycler, in accordancewith aspects of the present disclosure. Specifically, thermocycler 3500includes a plurality of inner segments 3502, 3504, 3506, 3508, 3510 thateach may be configured to define a separate temperature region. Thesesegments may be attached to a common core (not shown) or bound togetherin any suitable manner, and may be separated by air or any othersuitable medium, typically a thermally insulating material. The gapsbetween segments, if any, may have any chosen widths to generate adesired temperature profile in both the longitudinal direction and thetangential direction. As FIG. 92 depicts, the plurality of innersegments includes a different number of inner segments attached to thecore at different positions along the longitudinal axis.

Fluid traveling through fluidic tubing 3520 would encounter a firstportion 3512 of the thermocycler having just a single temperature regiondefined by segment 3502. Subsequently, the fluid would encounter asecond portion 3514 of the thermocycler having three temperature regionsdefined by segments 3504, 3506, and 3508. Next, the fluid wouldencounter a third portion 3516 of the thermocycler having twotemperature regions defined by segments 3504, 3508, and finally thefluid would encounter a fourth portion 3518 of the thermocycler having asingle temperature region defined by section 3510.

Any desired number of longitudinal portions, instead of or in additionto portions 3512, 3514, 3516 and 3518, may be included in athermocycler, to alter the number of temperature regions encountered bya fluid as it proceeds through a thermocycling process. Furthermore, anydesired number of tangential segments may be included within eachlongitudinal portion, so that particular windings of fluidic tubing maybe configured to encounter essentially any number of temperatureregions. By combining the features of thermocycler 3500 with thefeatures of thermocycler 3450 depicted in FIG. 91, a thermocycler can beconstructed to provide virtually any temporal temperature profile to amoving fluid, making the disclosed thermocyclers suitable for a widerange of applications.

F. Selected Embodiments 6

This example describes various additional aspects and possiblevariations of a thermocycler, in accordance with aspects of the presentdisclosure.

Whereas thermocyclers are primarily described above as including asingle “strand” of fluidic tubing wrapped substantially helically aroundthe circumference of heated sections of the thermocycler, manyvariations are possible. For example, more than one strand of tubing maybe provided, and the various strands all may be wrapped around a portionof the thermocycler. In some cases, the strands may be braided in somefashion so that they cross each other repeatedly, whereas in other casesthe strands all may be configured to directly contact the heatedthermocycler sections for substantially the entirety of their wrappedlength. In addition, one or more tubes may be configured to pass throughthe heated sections of a thermocycler, rather than wrapped around theirexteriors. For instance, the heated sections may be cast, molded, orotherwise formed around the tubes. In some cases, fluid tight channelsmay be formed in this manner, so that tubes are not necessary.

In some cases it may be desirable to vary the number of thermocyclesprovided by a thermocycling instrument, either dynamically or byproviding several varying options for the number of cycles a particularfluid will encounter. Dynamic changes in the number of thermocycles maybe provided, for example, by unwinding or additionally winding thefluidic tubing around the thermocycler. Optional numbers of cycles maybe provided, for example, by providing multiple fluidic tubes that arewound a different number of times around the instrument, or by creatingvarious optional bypass mechanisms (such as bypass tubes with valves) toselectively add or remove cycles for a particular fluid.

Although the heated segments of the thermocyclers described above aregenerally shown separated from each other by thermally insulating airgaps, any desired thermally insulating material may be placed betweenthe heated segments of a thermocycler according to the presentdisclosure. For example, the use of a low-density polymer or a silicaaerogel may provide increased thermal isolation of neighboring segments,both by reducing the thermal conductivity of the insulating regions andby decreasing convective heat transfer.

The disclosed thermocyclers may be used for PCR, any other molecularamplification process, or indeed any process involving cyclicaltemperature changes of a fluid sample, whether or not the sampleincludes discrete droplets. For example, potentially target-containingsamples may be separated into discrete units other than droplets, suchas by binding sample molecules to a carrier such as a suitable bead orpellet. These alternative carriers may be placed in a background fluidand thermocycled in much the same way as droplets in an emulsion.Alternatively, a plurality of thermocyclers may be used simultaneouslyto cycle different bulk fluid samples in parallel or in an overlappingsequence, without separating the fluid samples into many discrete units.

G. Selected Embodiments 7

This example describes additional aspects of a thermocycler, inaccordance with aspects of the present disclosure, presented withoutlimitation as a series of numbered sentences.

1. A method of thermocycling a sample-containing fluid to promote targetmolecule amplification, comprising (A) transferring an emulsion ofsample-containing droplets into a thermocycling instrument; (B)providing a denaturing temperature to the emulsion; (C) providing aprimer annealing temperature to the emulsion; and (D) providing apolymerase extension temperature to the emulsion; wherein providing thedenaturing temperature, the primer annealing temperature, and thepolymerase extension temperature respectively include transporting theemulsion in a substantially helical path cyclically through a denaturingtemperature region, a primer annealing temperature region, and apolymerase extension temperature region.

2. The method of paragraph 1, further comprising providing a hot-starttemperature to the emulsion, prior to providing the denaturingtemperature to the emulsion, by transporting the emulsion in asubstantially helical path through a hot-start temperature region.

3. The method of paragraph 1, wherein the temperatures are providedthrough the use of thermoelectric coolers configured to transfer heatbetween a thermal core and the temperature regions.

4. The method of paragraph 1, wherein the helical path decreases inlength over successive cycles.

5. A thermocycling system configured to promote molecular amplification,comprising (A) a core defining a central longitudinal axis; (B) aplurality of inner segments attached to the core and defining aplurality of temperature regions; (C) a plurality of heating elementsconfigured to maintain each temperature region approximately at adifferent desired temperature; and (D) a fluid channel configured totransport an emulsion of sample-containing droplets cyclically throughthe temperature regions.

6. The system of paragraph 5, further comprising a plurality of outersegments attached to the inner segments, and wherein the fluid channelis disposed between the inner and outer segments.

7. The system of paragraph 5, wherein the fluid channel is configured totransport the emulsion in a substantially helical path.

8. The system of paragraph 5, wherein the fluid channel includes fluidictubing wrapped around the inner segments.

9. The system of paragraph 8, wherein the fluidic tubing is disposed ingrooves of the inner segments that define a substantially helical patharound the inner segments.

10. The system of paragraph 5, wherein the fluid channel is disposedwithin the inner segments.

11. The system of paragraph 5, wherein the inner segments includeexternal grooves, and wherein the fluid channel is defined by thegrooves and by a fluid tight sheet wrapped around the inner segments.

12. The system of paragraph 5, wherein the core is configured as a heatsource and as a heat sink, and wherein the heating elements include atleast one thermoelectric cooler disposed between one of the innersegments and the core.

13. The system of paragraph 12, wherein at least one independentlycontrollable thermoelectric cooler is disposed between each of the innersegments and the core.

14. The system of paragraph 12, wherein the core is maintained at anoperating temperature that falls between two of the desiredtemperatures.

15. The system of paragraph 12, wherein the at least one thermoelectriccooler is disposed between a pair of thermally conductive andmechanically compliant pads.

16. The system of paragraph 5, wherein the core is unheated, and furthercomprising a thermal insulating layer disposed between the core and eachinner segment.

17. The system of paragraph 5, wherein the core includes a plurality ofcore sections, each independently in thermal contact with one of theinner segments.

18. The system of paragraph 5, wherein at least a portion of each innersegment is angled along the longitudinal axis so that the inner segmentscollectively form an approximately frustoconical shape.

19. The system of paragraph 5, wherein the plurality of inner segmentsincludes a different number of inner segments attached to the core atdifferent positions along the longitudinal axis.

20. A thermocycling instrument configured to promote molecularamplification, comprising (A) a core including a central aperturedefining a central longitudinal axis; (B) a plurality of inner segmentsattached to the core and defining a plurality of temperature regions;(C) a plurality of heating elements configured to maintain eachtemperature region approximately at a different desired temperature; (D)a fluid channel configured to transport an emulsion of sample-containingdroplets cyclically through the temperature regions; and (E) a thermallyconductive heat sink including a central aperture aligned with thecentral aperture of the core.

21. The instrument of paragraph 20, further comprising a cooling fanconfigured to blow air through the central aperture of the heat sink andthe central aperture of the core.

22. An apparatus for performing reactions in droplets, comprising (A) adroplet generator that produces droplets disposed in an immisciblecarrier fluid; (B) a heater assembly comprising at least twotemperature-controlled zones maintained at respective distincttemperatures (C) a coiled tube that receives droplets from the dropletgenerator and that traverses the temperature-controlled zones seriallyand repeatedly; and (D) a pump that drives travel of droplets throughthe coiled tube such that the droplets are cyclically heated and cooledby the temperature-controlled zones.

23. The apparatus of paragraph 22, wherein the distinct temperature ofat least one of the temperature-controlled zones is regulated by athermoelectric cooler.

24. The apparatus of paragraph 22, further comprising a controller incommunication with the thermoelectric cooler and programmed to activelyadjust electrical power supplied to the thermoelectric cooler tomaintain a set point temperature of at least one of thetemperature-controlled zones under varying thermal loads.

25. The apparatus of paragraph 22, wherein a pair of thetemperature-controlled zones are thermally coupled to each other by athermoelectric cooler.

26. The apparatus of paragraph 4, wherein the thermoelectric cooler isdisposed between the pair of temperature-controlled zones.

27. The apparatus of paragraph 22, wherein the heater assembly includesa thermally conductive core, and wherein each of thetemperature-controlled zones includes a conductive segment disposed atleast generally radially from the thermally conductive core.

28. The apparatus of paragraph 22, wherein the distinct temperature ofeach member of a pair of the temperature-controlled zones is regulatedby a respective thermoelectric cooler, and wherein the heater assemblyincludes a thermally conductive core that is connected to the respectivethermoelectric coolers and is maintained at a temperature intermediateto the distinct temperatures of the pair of temperature-controlledzones.

29. The apparatus of paragraph 22, wherein the tube wraps around theheater assembly a plurality of times.

30. The apparatus of paragraph 22, wherein the heater assembly includesa thermally conductive core and a heating element coupled to thethermally conductive core.

31. The apparatus of paragraph 22, wherein the heater assembly comprisesat least three temperature-controlled zones maintained at three or morerespective distinct temperatures, wherein the coiled tube comprises aplurality of coils, and wherein each coil thermally couples to each ofthe at least three temperature-controlled zones.

32. The apparatus of paragraph 31, wherein two or more coils of thecoiled tube thermally couple to a same temperature-controlled zone at asame range of angular positions on each of the coils.

33. The apparatus of paragraph 22, further comprising one or more otherdiscrete, coiled tubes that traverse the temperature-controlled zonesserially and repeatedly.

34. The apparatus of paragraph 33, wherein the at least one other coiledtube is interspersed with the coiled tube.

35. The apparatus of paragraph 22, further comprising at least onethermally controlled incubation region maintained at a predefinedincubation temperature, the incubation region being located upstreamfrom the temperature-controlled zones thereby causing a temperature ofdroplets flowing through the tube to at least substantially reach theincubation temperature prior to being heated and cooled cyclically bythe temperature-controlled zones.

36. The apparatus of paragraph 35, wherein heat for the incubationregion is supplied by a heater or a thermoelectric cooler.

37. An apparatus for performing reactions in droplets, comprising (A) aheater assembly comprising at least two temperature-controlled zonesmaintained at respective distinct temperatures, a temperature of atleast one of the temperature-controlled zones being regulated by athermoelectric cooler; (B) a coiled tube that traverses the temperaturezones serially and repeatedly; and (C) a pump that drives fluid flowthrough the coiled tube such that the fluid is cyclically heated andcooled by the temperature-controlled zones.

38. The apparatus of paragraph 37, wherein a pair of thetemperature-controlled zones are thermally coupled to each other by thethermoelectric cooler.

39. The apparatus of paragraph 38, wherein the thermoelectric cooler isdisposed between the pair of temperature-controlled zones.

40. The apparatus of paragraph 37, wherein the heater assembly includesa thermally conductive core, and wherein each of thetemperature-controlled zones includes a conductive segment disposed atleast generally radially from the thermally conductive core.

41. The apparatus of paragraph 37, wherein the distinct temperature ofeach member of a pair of the temperature-controlled zones is regulatedby a respective thermoelectric cooler, and wherein the heater assemblyincludes a thermally conductive core that is connected to each of therespective thermoelectric coolers and is maintained at a temperatureintermediate to the distinct temperatures of the pair oftemperature-controlled zones.

42. The apparatus of paragraph 37, wherein the tube wraps around theheater assembly a plurality of times.

43. The apparatus of paragraph 42, wherein the heater assembly includesa thermally conductive core and a heating element coupled to thethermally conductive core.

44. The apparatus of paragraph 37, wherein the heater assembly comprisesat least three temperature-controlled zones maintained at three or morerespective distinct temperatures, wherein the coiled tube forms aplurality of coils, and wherein each coil thermally couples to each ofthe at least three temperature-controlled zones.

45. The apparatus of paragraph 44, wherein two or more coils of thecoiled tube thermally couple to a same temperature-controlled zone at asame range of angular positions on each of the coils.

46. The apparatus of paragraph 37, further comprising one or more otherdiscrete, coiled tubes that traverse the temperature-controlled zonesserially and repeatedly.

47. The apparatus of paragraph 37, further comprising at least onethermally controlled incubation region maintained at a predefinedincubation temperature, the incubation region being located upstreamfrom the temperature-controlled zones thereby causing a temperature ofdroplets flowing through the tube to at least substantially reach theincubation temperature prior to being heated and cooled cyclically bythe temperature-controlled zones.

48. The apparatus of paragraph 47, wherein heat for the incubationregion is supplied by a heater or a thermoelectric cooler.

49. A method of nucleic acid analysis, comprising (A) generatingdroplets disposed in an immiscible carrier fluid, each droplet includinga partition of a sample disposed in an amplification reaction capable ofamplifying a nucleic acid target, if present in the droplet; (B) drivingthe droplets through a coiled tube that traverses two or moretemperature-controlled zones serially and repeatedly, to thermally cyclethe droplets under conditions promoting amplification of the nucleicacid target; (C) detecting one or more signals from one or more of thedroplets; and (D) determining a presence of the nucleic acid target inthe sample based on the signals.

50. A thermocycling apparatus comprising a coiled tube traversing aplurality of temperature controlled regions in at least onesubstantially helical winding, each of the regions including at least afirst zone maintained at a first temperature and a second zonemaintained at a second temperature thereby causing the temperature ofone or more droplets in an immiscible carrier fluid flowing through thetube to cycle between the first and the second temperatures.

51. The apparatus of paragraph 50, wherein the plurality of regionsincludes between two and fifty regions.

52. The apparatus of paragraph 50, wherein the temperature of at leastone of the temperature controlled zones is regulated by a thermoelectriccontroller.

53. The apparatus of paragraph 52, wherein at least two temperaturecontrolled zones are separated by a thermoelectric controller.

54. The apparatus of paragraph 50, wherein the temperature of the firsttemperature controlled zone is regulated by a first thermoelectriccontroller and the temperature of the second temperature controlledzones is regulated by a second thermoelectric controller.

55. The apparatus of paragraph 53, wherein the first and secondthermoelectric controllers are connected to a common conductor, andwherein the common conductor is maintained at a temperature intermediateto the first and second zone temperatures.

56. The apparatus of paragraph 50, wherein the droplets include at leastone of water, salt, DNA, RNA, proteins, prions, fluorescent dyes,probes, primers, surfactants sample, and nucleotides.

57. The apparatus of paragraph 50, wherein the immiscible carrier fluidincludes at least one of vegetable oil, fluorocarbon oil, mineral oil,and surfactants.

58. The apparatus of paragraph 50, wherein the coiled tube comprises aplurality of loops, and wherein the first and the second temperaturecontrolled zones extend across at least two of the loops, therebycausing the temperature of fluid flowing through the tube to cyclebetween the first and the second cycling temperatures at the samerelative angular position on each of the loops.

59. The apparatus of paragraph 58, wherein each winding comprises aplurality of separately controlled temperature controlled regions andthe temperature of any of the first and second zones of any member ofthe plurality of regions can be maintained at the same temperaturethereby allowing the angular section of the winding regulated at thefirst temperature and the angular section of the winding regulated atthe second temperature to be set to independent predetermined values.

60. The apparatus of paragraph 50, wherein the coiled tube furthercomprises at least one thermally controlled incubation region maintainedat a predefined incubation temperature, the incubation region locatedupstream from the temperature controlled regions thereby causing thetemperature of the fluid flowing through the tube to reach theincubation temperature prior to entering the cycling regions.

61. The apparatus of paragraph 60, wherein the heat for the incubationregion is supplied by either a thermoelectric controller or a resistiveheater.

62. The apparatus of paragraph 50, wherein the heat to maintain thetemperatures of the temperature controlled regions is provided by atleast one of conduction, convection, radiation, electric heaters,circulating liquid heaters, air blowers, incandescent light sources,lasers, LEDs, and microwaves.

63. The apparatus of paragraph 52, wherein the thermoelectric controlleris actively adjusted to maintain a substantially constant temperatureunder varying thermal loads caused by changes in advective heat flux,including at least one of the following changes: turning fluid flow onand off within the tube, changing flow rate of a fluid within the tube,alternating oil and droplet packets within the tube, receiving a plug ofcleaning solution within the tube, a change in density of fluid withinthe tube, a change in heat capacity of fluid within the tube, a changein thermal conductivity of fluid within the tube, and a change inthermal diffusivity of fluid within the tube.

64. An apparatus for performing a continuous-flow reaction, comprising(A) at least one capillary tube having a first open end for fluid inletand a second open end for fluid outlet to permit a continuous flow; and(B) at least two solid heating blocks, wherein the temperature of atleast one heating block is controlled by a thermoelectric controller.

65. The apparatus of paragraph 64 wherein at least one heating block iscontrolled by a resistive heater.

66. The apparatus of paragraph 64 wherein the heating blocks are indirect contact with each other.

67. The apparatus of paragraph 64 wherein the heating blocks aremaintained at different temperatures.

68. The apparatus of paragraph 64 wherein the apparatus comprises threeheating blocks, wherein a first heating block is maintained at atemperature between 85 and 99° C., a second heating block is maintainedat a temperature between 50 and 65° C., and a third heating block ismaintained at a temperature between 60 and 80° C.

69. The apparatus of paragraph 64 wherein the capillary tube is loopedaround the heating blocks.

70. The apparatus of paragraph 64 wherein the capillary tube contactsthe heating blocks sequentially and repetitively.

71. The apparatus of paragraph 64 wherein the capillary tube contactseach heating block at least 20 times.

72. An apparatus for performing a continuous-flow reaction, comprising(A) at least one capillary tube having a first open end for fluid inletand a second open end for fluid outlet to permit a continuous flow; and(B) at least two solid heating blocks, wherein at least one heatingblock is resistively heated and the capillary tube is looped around theheating blocks.

73. An apparatus for performing high-throughput nucleic acidamplification, comprising (A) a microdroplet generator comprising anorifice, wherein the orifice connects a sample flow pathway to a tubecomprising an immiscible fluid; (B) at least one capillary tube having afirst open end for fluid inlet and a second open end for fluid outlet topermit a continuous flow; and (C) a thermal cycling device, wherein thedevice has a plurality of fixed heating blocks, wherein the capillarytube is looped around the heating blocks and contacts the heating blockssequentially.

VI. DETECTION

This Section describes exemplary detection systems, for example, fordetecting sample-containing droplets. The systems may involve sensing ordetecting the droplets themselves and/or contents of the droplets. Thedetection of droplets themselves may include determining the presence orabsence of a droplet (or a plurality of droplets) and/or acharacteristic(s) of the droplet, such as its size (e.g., radius orvolume), shape, type, and/or aggregation state, among others. Thedetection of the contents of droplets may include determining the natureof the contents (e.g., whether or not the droplet contains a sample(s))and/or a characteristic of the contents (e.g., whether or not thecontents have undergone a reaction, such as PCR, the extent of any suchreaction, etc.).

The detection of droplets and their contents, if both are detected, maybe performed independently or coordinately, in any suitable order. Forexample, the detection may be performed serially (one droplet at atime), in parallel, in batch, and so forth.

The detection of droplets and their contents may be performed using anytechnique(s) or mechanism(s) capable of yielding, or being processed toyield, the desired information. These mechanisms may include opticaltechniques (e.g., absorbance, transmission, reflection, scattering,birefringence, dichroism, fluorescence, phosphorescence, etc.),electrical techniques (e.g., capacitance), and/or acoustic techniques(e.g., ultrasound), among others. The fluorescence techniques, in turn,may include fluorescence intensity, fluorescence polarization (orfluorescence anisotropy) (FP), fluorescence correlation spectroscopy(FCS), fluorescence recovery after photobleaching (FRAP), total internalreflection fluorescence (TIRF), fluorescence resonance energy transfer(FRET), fluorescence lifetime, and/or fluorescence imaging, amongothers.

The remainder of this Section describes exemplary detection systems,including droplet sensors and reaction sensors. In these exemplarysystems, the droplet sensor may generate and detect scattered light, andthe reaction sensor may generate and detect fluorescence, among otherapproaches. These systems are described, for convenience, in the contextof a PCR reaction; however, the techniques apply more generally to anyreaction, such as a biochemical reaction, capable of generating, orbeing modified to generate, a detectable signal.

In an exemplary PCR assay (or other nucleic acid amplification assay),the sample is first combined with reagents in a droplet, and the dropletis then thermocycled to induce PCR. It may then be desirable to measurethe fluorescence of the droplets to determine which, if any, containedone or more target nucleotide sequences. This generally involvesilluminating the droplets with radiation at a wavelength chosen toinduce fluorescence, or a change in a characteristic of thefluorescence, from one or more fluorescent probes associated with theamplified PCR target sequence(s). For example, in an exemplaryfluorescence intensity assay, if a relatively large intensity offluorescence is detected, this indicates that PCR amplification of thetarget nucleotide occurred in the droplet, and thus that the target waspresent in that portion of the sample. Conversely, if no fluorescence ora relatively small intensity of fluorescence is detected, this indicatesthat PCR amplification of the target nucleotide did not occur in thedroplet, and thus that a target was likely not present in that portionof the sample. In other fluorescence-based embodiments, the extent ofreaction could be determined from a decrease in fluorescence intensity,instead of a decrease, and/or a change in one or more other fluorescenceparameters, including polarization, energy transfer, and/or lifetime,among others.

The following examples describe specific exemplary detection systems, inaccordance with aspects of the invention. Additional pertinentdisclosure may be found in the U.S. provisional patent applicationslisted above under Cross-References and incorporated herein byreference, particularly Ser. No. 61/277,203, filed Sep. 21, 2009.

A. Example 1: Detection System 1

This example describes an optical detection system based on measuringthe end-point fluorescence signal of each sample/reagent droplet after aPCR amplification process is complete. The exemplary system is suitablefor making both qualitative and quantitative measurements; see FIGS. 93and 94.

FIG. 93 depicts a cytometry-type optical detection system, generallyindicated at 4200. The term “cytometry” refers to the fact that thedetection system is configured to detect both scattered and fluorescenceradiation. Detection system 4200 includes a radiation source 4202,transmission optics generally indicated at 4204, a forward scatterdetector 4206, and a fluorescence detector 4208. The forward scatterdetector may be replaced or augmented, in some embodiments, by sideand/or back scatter detectors, among others, configured to detect lightdetected to the side or back of the sample, respectively. Similarly, thefluorescence detector may be replaced or augmented, in some embodiments,by an epi-fluorescence detector, among others, configured to detectfluorescence emitted anti-parallel to the excitation light (e.g., backtoward transmission optics 4204 (which could, in such embodiments,include a dichroic or multi-dichroic beam splitter and suitableexcitation and emission filters)).

Sample-containing droplets 4210, which have already undergone at leastsome degree of PCR thermocycling, are transferred through a capillarytube or other similar fluid channel 4212, which intersects the path ofradiation from radiation source 4202 at an intersection region generallyindicated at 4214. An optical element 4216, such as a converging lens,may be placed between intersection region 4214 and forward scatterdetector 4206, to focus scattered radiation on the scatter detector.Similarly, an optical element 4218 may be placed between intersectionregion 4214 and fluorescence detector 4208, to focus fluorescenceradiation on the fluorescence detector. The system may include anobscuration bar 4219, operatively positioned between the sample anddetector, which reduces the amount of direct (unscattered) excitationradiation (light) that falls on the detector. The obscuration bar, shownhere as a small square object in front of optical element 4216, maycreate a triangular-shaped shadow 4219 a behind the optical element.This arrangement makes it easier for detector 4206 to detect changes inindex of refraction that have scattered (at small angles) the normalbeam.

Radiation from source 4202 may be partially scattered when it encountersa droplet, and the scattered radiation may be used to determine one ormore properties of the droplet. For example, scattered radiationindicating the presence of a droplet in intersection region 4214 may besensed by scatter detector 4206, and this information may be used toactivate fluorescence detector 4208, which may otherwise remaindeactivated (i.e., when a droplet is not present in the intersectionregion) to conserve power within the system. Even if the fluorescencedetector remains continuously active, detecting the presence of adroplet may be useful for other purposes. For example, tracking thedroplets passing through intersection region 4214 may be desirablebecause some droplets passing through the intersection region may not bedetected by the fluorescence detector (e.g., if the droplets do notcontain reaction product). In addition, tracking the droplets may allowbackground noise (i.e., the signal received by the detector in theabsence of a droplet) to be removed, improving the signal-to-noiseratio. Furthermore, as described below, various properties of a detecteddroplet may be estimated from data sensed by forward or side scatterdetector 4206.

Radiation detected by scatter detector 4206 may be used to infer thesize (or other properties) of a detected droplet. Specifically, ameasurement of the duration of a scattering event representing thepresence of a droplet within intersection region 4214, in conjunctionwith knowledge of the average speed of droplet passage through theintersection region, can be used to determine the width of the dropletin a plane normal to the direction of the incident radiation from source4202. If this width is less than the diameter of channel 4214, then itcan be inferred that the droplet is an approximate sphere with adiameter less than the diameter of channel 4214, and the volume of thedroplet can be calculated. If, on the other hand, the width of thedroplet exceeds the diameter of channel 4214, this indicates that thedroplet is likely contacting the walls of the channel and is notspherical. However, the droplet volume still may be estimated bymodeling the droplet as a cylinder or other similar shape passingthrough the channel. As described below, a determination of dropletvolume may be useful for normalizing the results of any correspondingfluorescence detection.

In some cases, radiation from source 4202 also may be scattered fromintersection region 4214 even if it does not encounter a droplet, forinstance, if it encounters a partially reflective surface such as afluid interface or a wall of fluid channel 4212. This type of scatteredradiation will generally have a different signature than radiationscattered from a droplet, so that it generally serves merely as abackground for droplet scattering events. Whether scattering occurs inthe absence of a droplet depends on the particular configuration ofsystem 4200, as will be described below. Similarly, scattering may occurwhen droplets outside a desired size range pass through the intersectionregion, and the signature of radiation scattered from such droplets maybe used to affect the subsequent treatment of such droplets. Forexample, the fluorescence signals received from unusually small or largedroplets may be removed from a statistical sample, to increasestatistical accuracy. In any case, after passing through intersectionregion 4214, scattered and/or unscattered radiation from radiationsource 4202 is directed toward forward scatter detector 4206.

Radiation from source 4202 that is absorbed by droplets withinintersection region 4214 may stimulate the emission of fluorescenceradiation that can be detected by fluorescence detector 4208. Morespecifically, radiation intersecting a droplet may excite a fluorescentprobe, such as a TAQMAN probe, that is configured to fluorescesignificantly only if the fluorescent portion of the probe becomesseparated from a quencher molecule. This separation, or cleaving,typically occurs only when polymerase replicates a sequence to which theprobe is bound. In other words, a probe will fluoresce significantlyonly in droplets within which a target nucleotide sequence has beenamplified through PCR. Accordingly, radiation source 4202 will generallybe configured to emit radiation at a wavelength that stimulatesfluorescent emission from one or more probes known to be present in thesample, and fluorescence detector 4208 will be configured to detect suchstimulated radiation.

Radiation source 4202 may take any form suitable for transmittingradiation at one or more desired wavelengths or wavelength bands. Forexample, radiation source 4202 may be a laser, such as a diode laser,emitting substantially monochromatic light at a wavelength of 488nanometers (nm) or at some other desired wavelength. Radiation source4202 also may include multiple separate lasers, emitting light at eithera single wavelength or at multiple different wavelengths. One or more(or all) of the lasers of radiation source 4202 may be replaced by analternate source of light, such as a light-emitting diode (LED)configured to emit a directed beam of radiation at one or more desiredwavelengths. In yet other embodiments, white light illumination, forexample, from a Halogen lamp, may also be used to provide the radiationsource.

Transmission optics 4204 may include any optical components suitable fordirecting, focusing, or otherwise desirably affecting radiation fromsource 4202. For example, as depicted in FIG. 93, the transmissionoptics may include one or more steering mirrors 4220, each configured todirect incident radiation in a desired direction such as toward anotheroptical component or toward intersection region 4214. Also as depictedin FIG. 93, the transmission optics may include a converging lens 4222,which is configured to focus radiation from source 4202 ontointersection region 4214 to maximize scattering and fluorescence causedby the radiation. The transmission optics may further include additionalcomponents such as aperture stops, filters, diverging lenses, shapedmirrors, and the like, to affect the transmission path and/or propertiesof the radiation from source 4202 before it arrives at intersectionregion 4214. In addition, the transmission optics further may include(in this and other embodiments) a mechanism for monitoring properties ofthe incident (excitation) radiation. For example, the transmissionoptics may include a partial mirror 4224 for reflecting a portion of theincident radiation to a detector 4226, such as a photodiode, formonitoring the intensity of the incident light. This would allowcorrection of the detected scattering and fluorescence for changes thatsimply reflect changes in the intensity of the incident light.

Forward scatter detector 4206 is configured to receive and detectradiation scattered from droplets passing through intersection region4214, as described previously. Various types of detectors may besuitable, depending on the desired cost and/or sensitivity of thedetector. In approximate order of decreasing sensitivity, exemplarytypes of scatter detectors that may be suitable include photodiodes,avalanche photodiodes, multi-pixel photon counters, and photomultipliertubes. The presence of optical element 4216, which typically will be aconverging lens used to refocus scattered radiation toward scatterdetector 4206, may decrease the necessary sensitivity of the forwardscatter detector for a given application, by increasing the intensityper unit area of scattered radiation incident on the detector.

Fluorescence detector 4208 is configured to receive and detectfluorescence radiation emitted by droplets at or near the time they passthrough intersection region 4214. Various types of fluorescencedetectors may be suitable, depending on factors such as desired costand/or sensitivity, including photodiodes, avalanche photodiodes,multi-pixel photon counters, and photomultiplier tubes. Also as in thecase of the forward scatter, utilizing an optical element 4218,typically a converging lens, between intersection region 4214 andfluorescence detector 4208 may decrease the necessary sensitivity of thefluorescence detector by increasing the intensity per unit area offluorescence radiation incident on the detector.

FIG. 94 depicts exemplary fluorescence measurements made by fluorescencedetector 4208. More specifically, FIG. 94 shows a post-PCR end-pointfluorescence trace from droplets, in which each “peak” 4230 representsthe intensity of detected fluorescence emitted by an individual dropletflowing through intersection region 4214. As FIG. 94 indicates, theresulting histogram can be used to identify positive from negativesignals. Specifically, the signals depicted in FIG. 94 each may becompared to a cut-off or threshold fluorescence level, as indicated bydashed line 4232. Signals exceeding the threshold level will beinterpreted as positive for PCR amplification, and thus for the presenceof the target nucleotide sequence in the corresponding droplet, asindicated for an exemplary signal at 4234. On the other hand, signalsfalling below threshold level 4232 will be interpreted as negativeoutcomes, indicating that the corresponding droplet did not contain thetarget.

An example of a negative signal is indicated at 4236, where thedetection of a sub-threshold amount of fluorescence is due to thepresence of uncleaved fluorescent probe in the droplet. As describedpreviously, the fluorescence of such probes is generally not completelyquenched even in the absence of cleavage by a binding polymerase. Also,the differences in fluorescent intensity of a positive, as seen in thesignal voltage peak heights between the positive peak at 4230 andpositive peak 4234, can be attributed to different amounts of startingnucleic acid target originally in the droplet prior to PCR (e.g., oneversus two starting targets). The ratio of different amounts of startingtarget amounts may be governed by Poisson statistics.

Typically, hundreds to millions of droplets are analyzed per run. In anycase, after a desired number of signals have been detected byfluorescence detector 4208, i.e., after a desired number of dropletshave passed through intersection region 4214, the positive and negativesignals are counted and analyzed. Analysis is typically performed usingreceiver-operator characteristic curves and Poisson statistics todetermine target presence and target concentration, respectively.Running analysis using Poisson statistics can also be performed to givean estimate of target concentration prior to processing all the droplets(i.e., subsets of the total droplets are used in the statisticalanalysis). The analysis of droplets is further described in Section VII.

B. Example 2: Detection Systems Using Optical Fibers

This example describes fluorescence detectors configured to measure theend-point fluorescence signal of sample/reagent droplet after PCR, andwhich utilize one or more optical fibers to transmit radiation to and/orfrom an intersection region within which illuminating radiationintersects the path of the sample-containing droplets. The exemplarysystems are suitable for making both qualitative and quantitativemeasurements; see FIGS. 95-99.

FIG. 95 depicts an optical detection system, generally indicated at4250, which is similar to system 4200 depicted in FIG. 93 except thattransmission optics 4204 of system 4200 have been replaced by an opticalfiber 4254. Optical fiber 4254 may be constructed from a glass, aplastic, and/or any other material that is substantially transparent toradiation of one or more particular desired wavelengths and configuredto transmit that radiation along the length of the fiber, preferablywith little or no loss of intensity.

Replacing the transmission optics with optical fiber 4254 may allowsystem 4254 to be constructed relatively inexpensively and in a morespace-saving manner than systems using optical elements such as mirrorsand lenses. This results from the fact that the cost and spaceassociated with the other optical elements is no longer necessary, andalso from the fact that optical fiber 4254 may be shaped in any desiredmanner, allowing significant design flexibility. Aside from opticalfiber 4254, detection system 4250 otherwise includes a radiation source4252, a forward scatter detector 4256, and a fluorescence detector 4258,all of which are similar to their counterparts in system 4200 and willnot be described again in detail.

Optical fiber 4254 is depicted in FIG. 95 as ending a short distancefrom droplets 4260 traveling in fluid channel 4262 through anintersection region generally indicated at 4264, in which radiationemitted from the end of the optical fiber intersects with the dropletstraveling through the fluid channel. Other configurations are possiblein which, for example, the optical fiber is configured to focusradiation more precisely toward the intersection region and/or isintegrated directly into the fluid channel. These possibilities aredescribed below in more detail; see FIGS. 98 and 99 and accompanyingdiscussion.

FIG. 96 depicts an optical detection system, generally indicated at4270, which is similar to system 4200 depicted in FIG. 93 except thatoptical elements 4216 and 4218 of system 4200 have been replaced byoptical fibers 4286 and 4288 in system 4270 of FIG. 96. As in the caseof optical fiber 4254 shown in FIG. 95 and described above, opticalfibers 4286 and 4288 each may be constructed from a glass, a plastic,and/or any other material that is substantially transparent to radiationof one or more particular desired wavelengths and configured to transmitthat radiation along the length of the fiber, preferably with little orno loss of intensity.

In the case of system 4270, optical fiber 4286 will be configured totransmit at least scattered radiation having a wavelength equal to thewavelength of light emitted by radiation source 4272 (which generallydoes not change during scattering), and optical fiber 4288 will beconfigured to transmit at least fluorescence radiation emitted by anyfluorescent probes within droplets 4280 that are excited by incidentradiation from source 4272. Accordingly, optical fibers 4286 and 4288may in some cases be constructed from different materials. The use ofoptical fibers 4286 and 4288 may result in cost and space savings forthe same reasons described previously with respect to the use of opticalfiber 4254 in system 4250.

Aside from the use of optical fibers 4286 and 4288, system 4270 issimilar to system 4200, including radiation source 4272, transmissionoptics 4274, a forward scatter detector 4276, and a fluorescencedetector 4278, which are similar to their previously describedcounterparts and will not be described further. Radiation from source4272 passes through transmission optics 4274 and encounters droplets4280 traveling through fluid channel 4282, at an intersection region4284. Some of the forward scattered radiation is transmitted throughoptical fiber 4286 to forward scatter detector 4276. Similarly, some ofthe fluorescence radiation emitted from droplets 4280 is transmittedthrough optical fiber 4288 to fluorescence detector 4278. As in the caseof optical fiber 4254 in FIG. 95, optical fibers 4286 and 4288 are shownstarting at a distance from fluid channel 4282, but as noted above,other configurations are possible and will be described below withreference to FIGS. 98 and 99.

FIG. 97 depicts an optical detection system, generally indicated at4300, in which optical fibers are used to transmit both incident andoutgoing radiation. More specifically, system 4300 includes a radiationsource 4302, an optical fiber 4204 for transmitting emitted radiationaway from source 4302, a forward scatter detector 4306, and afluorescence detector 4308. Post-PCR sample-containing droplets 4310 aretransferred through fluid channel 4312 toward intersection region 4314.Optical fiber 4316 transmits scattered radiation from intersectionregion 4314 to forward scatter detector 4306, and optical fiber 4318transmits fluorescence radiation from intersection region 4314 tofluorescence detector 4308.

As described previously, the use of optical fibers may result in variouscost and space savings. These savings may be further amplified, relativeto systems 4250 and 4270, by the use of fiber optics for all of theradiation transfer in system 4300. Aside from the use of optical fibersfor radiation transfer and any associated efficiencies, system 4300 issimilar in both its components and its operation to the previouslydescribed systems, and accordingly will not be described further.

FIG. 98 shows a magnified view of an intersection region, generallyindicated at 4320, in which incident radiation from a radiation source(not shown) is transmitted through an optical fiber 4322 to intersectwith sample-containing droplets 4324 traveling through a droplet inputfluid channel 4326. Intersection region 4320 differs from theintersection regions described previously in that optical fiber 4322 isintegrated into a radiation input fluid channel 4328 that is fluidicallyconnected with fluid channel 4326. Thus, radiation is emitted fromoptical fiber 4322 directly into the fluid within the connected fluidchannels, so that it encounters droplets 4324 without crossing either aninterface between air and the fluid channel material (typically someform of glass) or an interface between the fluid channel material andthe fluid within the channel.

By configuring the intersection region in this manner and avoiding twointerfaces between media with different indices of refraction,undesirable reflections of the incident radiation may be decreased,resulting in a greater intensity of radiation reaching droplets 4324.Furthermore, embedding optical fiber 4322 within a connected fluidchannel may allow for more convenient and stable placement of theoptical fiber at a small distance from fluid channel 4326 and at adesired orientation relative to fluid channel 4326, again potentiallyresulting in a greater intensity of radiation reaching the droplets. Tosecure optical fiber 4322 in place within channel 4328, a fluidicfitting 4330 may be placed at an end of channel 4328, and configured sothat optical fiber 4322 passes through an aperture of the fitting in afluid tight manner.

Intersection regions of the type depicted in FIG. 98 may take variousforms. For example, as depicted in FIG. 98, optical fiber 4322 may havea slightly smaller outer diameter than the inner diameter of fluidchannel 4328. Alternatively, optical fiber 4322 may have an outerdiameter approximately equal to the inner diameter of fluid channel4328, which may lead to an even more secure placement of the opticalfiber within the fluid channel. In addition, FIG. 98 depicts an outgoingoptical fiber 4332 disposed within a fluid channel 4334 that is alsofluidically connected with fluid channel 4326. Optical fiber 4332, whichis secured within channel 4334 by a fluidic fitting 4336, is configuredto transmit scattered radiation to a forward scatter detector (notshown). In some embodiments, one of incoming optical fiber 4322 andoutgoing optical fiber 4332 may be used, but not the other. Furthermore,one or more additional optical fibers, such as an outgoing optical fiberleading to a fluorescence detector (not shown) may be fluidicallycoupled into intersection region 4320.

FIG. 99A depicts another intersection region, generally indicated at4340, between sample-containing droplets 4342 traveling through a fluidchannel 4344 and excitation radiation 4346 emitted from a radiationsource (not shown). Excitation radiation 4346 is transmitted tointersection region 4340 through an optical fiber 4348, which isoriented with its long axis parallel to fluid channel 4344. As depictedin FIG. 99A, optical fiber 4348 may come to a point or otherwise betapered in the region proximal to fluid channel 4344, to focusexcitation radiation 4346 (through internal reflections within theoptical fiber) into channel 4344 and toward droplets 4342. This mayallow the excitation radiation to be directed primarily at a singledroplet 4342′, despite the collinear disposition of optical fiber 4348with multiple droplets.

Fluid channel 4344, which is configured to transport the droplets tointersection region 4340 where the droplets encounter stimulatingradiation transmitted by optical fiber 4348, is shown splitting into two(or more) outgoing fluid channels 4350 and 4352 after droplets 4342 passthrough the central part of intersection region 4340. This allows thesample-containing droplets to continue their motion through the PCRsystem while still allowing a collinear arrangement of fluid channel4344 and optical fiber 4348. As FIG. 99A illustrates, the outgoing fluidchannels and the optical fiber may be given complementary shapes, sothat the optical fiber fits snugly between outgoing channels 4350 and4352. This may lead to a relatively stable collinear configuration ofthe optical fiber and fluid channel 4344 (to help self-align the fiberand channel).

The intersection region shown in FIG. 99A is configured so that opticalfiber 4348 transmits both excitation radiation 4346 and alsofluorescence radiation 4354 emitted by the droplets. The fluorescenceradiation is then transmitted back through the optical fiber and towarda fluorescence detector (not shown), which may be integrated with aradiation source into a single component. Due to the shape of theproximal end of optical fiber 4348, emitted fluorescence radiation fromstimulated droplet 4342′ may enter optical fiber 4348 both “head on” andalso from a subsequent position along one side of the optical fiber.This effectively lengthens the integration time of the fluorescencedetection, resulting in better detection with a given detectorsensitivity.

FIG. 99B depicts another intersection region, generally indicated at4360, which is similar in some respects to intersection region 4340 ofFIG. 99A. Specifically, an optical fiber 4368 in FIG. 99B is configuredto transmit excitation radiation 4366 from a radiation source (notshown) toward sample containing droplets 4362 traveling in a fluidchannel 4364, and fluorescence radiation 4374 from an excited droplet4362′ back through the optical fiber and toward a fluorescence detector(not shown). Unlike intersection region 4340, however, fluid channel4364 of intersection region 4360 is oriented mostly perpendicular to thelong axis of optical fiber 4368, except for a “dog leg” or side-facingregion 4380 in the central portion of intersection region 4360.

Side-facing region 4380 of intersection region 4360, which is configuredto transport the droplets to intersection region 4360 where the dropletsencounter stimulating radiation transmitted by optical fiber 4368, isconfigured to allow only a small number of droplets, such as one dropletat a time, to travel parallel to the long axis of optical fiber 4368.This configuration may result in relatively more accurate detection offluorescence radiation, because only one droplet (or a small number ofdroplets) is stimulated with incident radiation at a time, and only thestimulated droplet(s) emits substantial fluorescence radiation back intooptical fiber 4368 for detection.

Optical fiber 4368 of FIG. 99B may be partially or completely surroundedby fluid, and this surrounding fluid may be in fluid communication withfluid channel 4364. However, unlike fluid channels 4350 and 4352 of FIG.99A, fluid regions 4370 and 4372 surrounding optical fiber 4368, whichmay in some cases constitute a single continuous fluid region, are toosmall to allow passage of any sample-containing droplets. Rather, thesesurrounding fluid region(s) are configured primarily to removeunnecessary interfaces between the optical fiber and the droplets,increasing the intensity of the incident radiation as describedpreviously.

C. Example 3: Detection Systems with Plural Radiation Channels

In some cases, a detection system according to the present disclosuremay include multiple separate incident radiation channels to illuminatesample-containing droplets that have undergone PCR thermocycling. Thisexample describes two such systems and some of their potential uses; seeFIGS. 100 and 101.

FIG. 100 depicts a multi-channel cytometry-type optical detectionsystem, generally indicated at 4400. Detection system 4400 includes aradiation source 4402, configured to emit radiation at one or moredesired wavelengths. As described previously, a radiation sourceaccording to the present disclosure may be of various types, such as anLED source or a laser source, and may emit radiation substantially at asingle wavelength, at a plurality of substantially discrete wavelengths,or within one or more ranges of wavelengths.

Radiation from source 4402 passes from the source toward transmissionoptics, as generally indicated at 4404. Transmission optics 4404 mayinclude one or more optical elements, such as a mirror 4406, configuredprimarily to redirect radiation emitted by source 4402 in a desireddirection. Transmission optics 4404 also may include one or more opticalelements, such as reflective elements 4408, 4410, 4412, configured tosplit the radiation emitted by source 4402 into several differentportions, each of which may be redirected in a particular manner, suchas the manner shown in FIG. 100. Alternatively, radiation source 4402may be omitted, and reflective elements 4408, 4410, 4412 each may bereplaced by a separate radiation source. In some cases, providing pluralradiation sources in this manner may be simpler than splitting theradiation from a single source.

In some instances, reflective elements 4408, 4410, 4412 may beconfigured to transmit and reflect incident radiation in different ways.For example, reflective element 4408 may be configured to reflectapproximately one-third of the radiation incident upon it and totransmit approximately two-thirds of the radiation incident upon it,reflective element 4410 may be configured to reflect approximatelyone-half of the radiation incident upon it and to transmit approximatelyone-half of the radiation incident upon it, and reflective element 4412may be configured to reflect substantially all of the radiation incidentupon it. In this manner, radiation emitted by radiation source 4402 maybe split into three portions of approximately equal intensity.

In cases where it is desirable to split the radiation emitted by source4402 into a number of channels other than three, a plurality ofreflective surfaces may be configured appropriately. Specifically, whenn channels are desired, n reflective elements may be used, with thefirst reflective element configured to reflect fraction 1/n and totransmit fraction (n−1)/n of the radiation incident upon it, the secondreflective element configured to reflect fraction 1/(n−1) and totransmit fraction (n−2)/(n−1) of the radiation incident upon it, thethird reflective element configured to reflect fraction 1/(n−2) and totransmit fraction (n−3)/(n−2) of the radiation incident upon it, and soforth, until the last reflective element in the sequence is a puremirror that reflects all of the radiation incident upon it and transmitsnone. This results in each of the n reflective elements reflecting anequal fraction 1/n of the radiation emitted by the radiation source.

An arrangement configured to split radiation from a source into severalportions of either approximately equal intensity or differingintensities may be useful, for example, when it is desirable to searchfor various targets, each of which is bound to a fluorescent probeconfigured to be excited by the same wavelength of incident radiationbut to fluoresce at a different wavelength. For instance, reflectivesurfaces 4408, 4410 and 4412 may be configured to reflect radiationtoward intersection regions 4414, 4416 and 4418, respectively, which maybe viewed as different adjacent portions of a single, largerintersection region. Similarly, when a plurality of radiation sourcesare used instead of reflective surfaces, each radiation source may beconfigured to transmit fluorescence stimulating radiation to a differentadjacent portion of the intersection region.

In the intersection region(s), the arriving radiation will intersect afluid channel 4420 (such as a capillary tube) through whichsample-containing droplets 4422 are moving. Each droplet thus may beirradiated a plurality of times, and accordingly may be stimulated toemit fluorescence radiation a plurality of times if the irradiateddroplet contains any of several desired target nucleic acid sequences.However, the droplet may emit a different wavelength of stimulatedradiation depending upon which target it contains (and thus whichfluorescent probe has been cleaved from its associated quenchingmolecule by replication of the target).

To detect stimulated fluorescence radiation corresponding to the varioustargets, a plurality of fluorescence detectors 4424, 4426, 4428 may beused, with each detector positioned and oriented to receive fluorescenceradiation produced at a different one of intersection regions 4414,4416, 4418 (or at a different portion of the larger intersection regionencompassing regions 4414, 4416, 4418). Furthermore, each fluorescencedetector may be configured to detect fluorescence at a differentwavelength, corresponding to one or more (but not all) of the varietiesof target molecules or target nucleic acid sequences. Thus, a givenirradiated droplet may emit stimulated fluorescence that is detected byjust one of detectors 4424, 4426, 4428, resulting in a “positive”detection of just one (or a subset) of the target sequences. In thismanner, system 4400 may be used to search for multiple targetssimultaneously.

Splitting incident radiation in the manner of system 4400 also may beuseful when it is desirable to illuminate sample-containing droplets formore time than it takes the droplet to pass through the unsplit beam ofthe source. For instance, as described above, system 4400 may beconfigured so that droplets 4422 passing through a fluid channel 4420intersect radiation from source 4402 at several intersection regions4414, 4416, 4418 corresponding to the various split beams. If theseintersection regions are disposed relatively near each other, then eachdroplet may essentially be continuously illuminated in an area spanningall of the intersection regions 4414, 4416, 4418. The resultingrelatively long integration time (i.e., the time of exposure of adroplet to illuminating radiation) may result in greater fluorescencefrom each target-containing droplet, and thus in greater accuracy of thedetection system. Another way to obtain a similar result is illustratedin FIG. 101 and will be described in detail below.

Still considering FIG. 100, detection system 4400 also may be used tosearch for multiple different nucleic acid targets in cases wherevarious probes that respond to different incident wavelengths ofexcitation radiation have been combined with a sample. For example,radiation source 4402 may be configured to emit radiation at a pluralityof discrete wavelengths or wavelength ranges, by using a plurality ofradiation emitters or a single emitter configured to produce radiationat all of the desired wavelengths. In this case, each of reflectivesurfaces 4408 and 4410 (and possibly 4412) may be dichroic andconfigured to reflect substantially all of the radiation at a particularwavelength (or within a particular wavelength range) and to transmit theremaining incident radiation. Alternatively, as described above, aplurality of radiation sources may be provided and configured totransmit fluorescence stimulating radiation at a different wavelength.

When dichroic reflective surfaces are provided, reflective surface 4408may be configured to reflect a particular wavelength or wavelength rangetoward intersection region 4414, reflective surface 4410 may beconfigured to reflect another particular wavelength or wavelength rangetoward intersection region 4416, and reflective surface 4412 may beconfigured to reflect yet another particular wavelength or wavelengthrange toward intersection region 4418. Alternatively, reflective surface4412 may be configured to reflect all radiation toward region 4418,since this will include any desired radiation that was not alreadyreflected by surfaces 4408 and 4410. Accordingly, different wavelengthsof incident radiation will arrive at each intersection region 4414,4416, 4418, and stimulated fluorescence emission will occur only if aprobe sensitive to a particular arriving wavelength has been activateddue to polymerase cleaving of its associated quenching molecule, i.e.,only if a particular target is present. Detectors 4424, 4426, 4428 maybe used to monitor the activation of droplets within the variousintersection regions, as described previously.

FIG. 101 depicts another multi-channel cytometry-type optical detectionsystem, generally indicated at 4450. System 4450 is generally similar tosystem 4400, including a radiation source 4452 and transmission opticsgenerally indicated at 4454. In the case of system 4450, thetransmission optics may include first and second mirrors 4456, 4458configured to redirect radiation emitted by source 4452 in a desiredmanner. Transmission optics 4454 also may include one or more otheroptical elements (not shown) for focusing radiation from source 4452, asdescribed previously.

As indicated in FIG. 101, mirror 4458 may be adjustable so that it isconfigured to reflect radiation at a range of different angles, todirect it toward a range of different positions along a fluid channel4460 through which sample-containing droplets 4462 are beingtransferred. Thus, the reflected radiation defines an intersectionregion, generally indicated at 4464, which is substantially wider thanit would be if mirror 4458 was fixed in a single orientation. If mirror4458 is adjusted relatively rapidly, this configuration may allowradiation from source 4452 to illuminate more than one droplet at atime, or may cause a single droplet to fluoresce at various positionswithin fluid channel 4460. In this case, a plurality of detectors 4466,4468, 4470 may be oriented to look for radiation at particularwavelengths corresponding to various target probes.

Alternatively, if the adjustment speed of mirror 4458 is chosen tocorrespond to the known approximate speed of sample-containing dropletstraveling within fluid channel 4460, then the mirror may effectivelyincrease the illumination time of each droplet by “tracking” the dropletthrough the channel. In this case, it may be appropriate to use only asingle fluorescence detector, with a field of view that spans the entirepath traveled by a droplet during its illumination.

D. Example 4: Separation of Droplets

This example describes fluid focus mechanisms for achieving a desiredseparation between sample-containing droplets as they pass through afluorescence detection system; see FIGS. 102-104. As the discussionabove indicates, it may be desirable for droplets within a detectionregion to be separated by some known average distance, or at least bysome approximate minimum distance. For example, adequate spacing maypermit split beams of radiation and/or detectors to be disposed mostappropriately, and may allow a suitable choice of adjustment range foran adjustable mirror, when one is used.

In addition, proper spacing can help to avoid unintentionally detectingradiation from two or more droplets simultaneously, which can result infalse positives and other errors in the detection system. For instance,as described previously, an uncleaved probe within a droplet still emitssome amount of fluorescence even though the nucleic acid target is notpresent in the droplet. Thus, the intensity of fluorescence emitted fromtwo or more droplets, neither of which contains a target, may besufficient to trigger a positive detection result if the fluorescencefrom those multiple droplets is mistakenly thought to come from a singledroplet. Other errors, such as errors in determining droplet volume andtarget concentration, also may result when droplets are spaced tooclosely together.

FIG. 102 shows a fluid focus mechanism, generally indicated at 4480,which is configured to separate sample-containing droplets from eachother by some desired amount of distance. This mechanism may be used,for example, to separate droplets prior to transferring them toward adetector intersection region such as intersection region 4214 of FIG.93, intersection region 4264 of FIG. 95, or any of the otherintersection regions described above. Focus mechanism 4480 includes adroplet input channel 4482, which contains sample-containing droplets4484 that are spaced closely together. Focusing fluid, indicated byarrows 4486, is transferred through focus fluid input channels 4488,4490, so that it encounters droplets from the droplet input channel at afocus region generally indicated at 4492.

A droplet entering focus region 4492 will be channeled into dropletegress channel 4494, which is the only channel through which fluid canexit the focus region. Egress channel 4494 may be configured to haveregions with a smaller inner diameter 4496 than the inner diameter ofsome or all of droplet input channel 4482 and focus fluid input channels4488, 4490, although in some instances this may not be the case. Becausefluid is flowing into focus region 4492 from focus fluid input channels4488 and 4490 as well as from droplet input channel 4482, and/or becauseegress channel 4494 has a smaller cross sectional area than the otherchannels, fluid will flow more rapidly through the egress channel thanthrough the other channels.

Because of the increase in fluid speed as fluid approaches the egresschannel, droplets will accelerate as they enter the egress channel, andwill become separated from each other as FIG. 102 indicates. Byappropriate choices of channel inner diameters and focus fluid inputvelocity, essentially any desired average spacing between droplets canbe achieved. Within egress channel 4494, there may be an irradiationzone, generally indicated at 4498. The irradiation zone may havefeatures, such as increased transparency and/or thinner channel walls,which are conducive to irradiating droplets with radiation from aradiation source 4500. A forward scatter detector 4502 and afluorescence detector 4504 may be positioned appropriately to detectscattered and fluorescence radiation, as described previously.

FIG. 103 shows another fluid focus mechanism, generally indicated at4510. As in the case of fluid focus mechanism 4480 of FIG. 102, fluidfocus mechanism 4510 is configured to increase the distance betweenclosely spaced sample-containing droplets to some desired minimumaverage value. Fluid focus mechanism 4510 includes a droplet inputchannel 4512 that has a body portion 4514 and a neck portion 4516. Bodyportion 4514 may be configured to contain a relatively large number ofclosely spaced sample-containing droplets 4515, as FIG. 103 depicts, orin some cases it may contain a stream of continuously flowing droplets.In either case, the diameter of neck portion 4516 may be chosen tosubstantially match, or to be just slightly larger than, the expectedaverage droplet diameter, so that only one droplet at a time willtypically be able to travel through the neck portion.

Mechanism 4510 also includes an outer fluid channel 4518, whichsurrounds at least a portion of droplet input channel 4512, includingneck portion 4516. In conjunction with droplet input channel 4512, outerfluid channel 4518 defines a focus fluid input channel 4520 between thedroplet input channel and the outer fluid channel. Typically, dropletinput channel 4512 and outer fluid channel 4518 will be cylindrical, sothat focus fluid input channel 4520 will take the form of a concentriccylindrical shell. Focusing fluid, generally indicated by arrows 4522,may be transferred through focus fluid input channel 4520 at a desiredvelocity. Accordingly, as each droplet 4515 exits neck portion 4516, itwill accelerate away from the neck portion due to the flow of thefocusing fluid. Through careful selection of the geometry of the systemand the focusing fluid velocity, any desired separation between adjacentdroplets exiting the neck portion can be attained. A radiation source4524, a forward scatter detector 4526, and a fluorescence detector 4528may be provided to irradiate, track, and analyze droplets as describedpreviously.

FIG. 104 is a section of fluidic tubing 4540 illustrating how anappropriate choice of fluid channel diameter(s) can contribute to anappropriate separation between droplets. This point was discussed above,in the description of neck portion 4516 of fluid focus mechanism 4510.This description applies not only to a neck portion of a droplet inputchannel, but also more generally to any fluid channel through whichdroplets pass within a detection system according to the presentdisclosure. For example, the same considerations apply to fluid channel4512 of FIG. 93, fluid channel 4262 of FIG. 95, etc.

As FIG. 104 depicts, fluidic tubing 4540 may be selected to have aninner diameter that is correlated with the expected average dropletdiameter. Accordingly, a droplet 4542 having a slightly smaller thanaverage diameter will be relatively unlikely to be in close proximity toadditional droplets in the tubing. Similarly, a droplet 4544 having theexpected average diameter will move freely within tubing 4540, and willmaintain its spherical shape. Finally, a droplet 4546 having a diameterslightly greater than the expected average diameter will take on apartially cylindrical shape, the volume of which may be estimatedaccordingly. Thus, an appropriate choice of fluid tubing size can helpto ensure proper separation between droplets.

E. Example 5: Batch Fluorescence Detection

In some cases, it may be desirable to irradiate and/or detectfluorescence from sample-containing droplets in relatively large batchesrather than one droplet at a time. This example describes a system fordetecting fluorescence emitted from a plurality of droplets that havebeen transferred to a chamber for batch detection; see FIG. 105.

FIG. 105 schematically depicts a batch optical detection system,generally indicated at 4560. In contrast to the previously describedcontinuous flow detection systems, in which sample-containing dropletsflow continuously through an intersection region where excitationradiation intersects the path of the moving droplets, system 4560 isconfigured to detect radiation from a plurality of droplets that havebeen collected in a detection region, and in some cases temporarilystopped from flowing through the system. This allows the fluorescencelevel of many droplets to be detected in a single detection operation,which may be advantageous in some applications.

Batch detection system 4560 includes a droplet input channel 4562,within which sample-containing droplets 4564 may be caused to flow in anemulsion (such as a water-in-oil emulsion), just as in the previouslydescribed detection systems. System 4560 also includes a valvemechanism, generally indicated at 4566, which is configured toselectively direct droplets toward either of two fluorescence detectionchambers 4568, 4570. For example, valve mechanism 4566 may include afirst valve 4572 disposed between droplet input channel 4562 anddetection chamber 4568, and a second valve 4574 disposed between dropletinput channel 4562 and detection chamber 4570. Thus, by opening andclosing valves 4572 and 4574 appropriately, droplets may be transferredselectively into chambers 4568, 4570. This may allow a substantiallycontinuous flow of emulsion to be transferred from the droplet inputfluid channel to the fluorescence detection chambers.

Chambers 4568, 4570 may be configured to have a relatively shallowdepth, to allow substantially only a monolayer of droplets within eachchamber, so that only one droplet is disposed within each portion of theline of sight of a detector and is confined to the focal plane of thedetector. Alternatively, various three-dimensional detectionconfigurations, such as confocal imaging or wide-field imaging withdeconvolution, may be used with non-monolayer samples.

A radiation source 4576 is configured to illuminate droplets withinchambers 4568, 4570, and after a desired number of droplets aretransferred into one of the detection chambers, the chamber may beilluminated with radiation from source 4576. Source 4576 may beconfigured in various ways to illuminate substantially all of thedroplets within a chamber. For example, radiation source 4576 mayinclude a single radiation emitting element, configured to illuminatesubstantially the entire chamber either by emitting a broad beam ofradiation or by emitting radiation toward intermediate optics (notshown) that spread the emitted beam to cover the entire chamber. Theradiation source also may include a plurality of radiation emittingelements, such as lasers, LEDs, and/or lamps, among others, eachconfigured to illuminate a portion of the appropriate detection chamber.Alternatively or in addition, one or more radiation emitting elements ofradiation source 4576 may be configured to scan the chamber, tosequentially illuminate droplets within the chamber, or the chamberitself may be configured to move so that all portions of the chamberintersect a substantially stationary beam of radiation. In some cases, acombination of two or more of the above techniques may be effective.

A fluorescence detector 4578 is provided and configured to detectfluorescence emitted from droplets 4564. As has been describedpreviously, the amount of fluorescence emitted by a particular dropletis expected to be significantly higher if the droplet contains a targetnucleotide sequence, because in that case the corresponding fluorescentprobe will typically have been cleaved from its associated quenchingmolecule. Thus, after the droplets within a detection chamber have beenilluminated with stimulating radiation or in some cases whileillumination is occurring, detector 4578 may be configured to receivefluorescence from the detection chamber. As in the case of illumination,detection may proceed in various ways. For example, a large formatdetector such as a CCD focal plane array may be used to detect radiationemitted from an entire detection chamber simultaneously. Alternatively,a smaller detector such as a photodiode or a photomultiplier may bescanned across the chamber, or the chamber may be repositioned withrespect to the detector, to detect fluorescence radiation from variousportions of the detection chamber sequentially.

System 4560 may be configured to allow substantially continuous flowthrough droplet input channel 4562, by transferring droplets into two ormore detection chambers, such as chambers 4568, 4570, sequentially. Forexample, FIG. 105 depicts the system at a time when chamber 4568 hasalready been filled with droplets and is being illuminated and/orimaged, whereas chamber 4570 is in the process of being filled.Accordingly, valve 4572 will be in its closed position, and valve 4574will be in its open position, to allow droplets to flow into chamber4570.

Upon completion of the detection process on the droplets within chamber4568, valve 4574 may be closed, valve 4572 may be opened, and anothervalve 4580 at the distal end of chamber 4568 also may be opened. Thisstops the flow of droplets into chamber 4570 and restarts the flow ofdroplets into chamber 4568, while allowing the droplets already inchamber 4568 to escape through distal valve 4580. Another distal valve4582 may be disposed at the end of chamber 4570 for a similar purpose.Alternatively, before the flow of droplets into a given chamber isresumed, and while droplets are still flowing into the other chamber,the chamber not receiving droplets may be washed with a fluid thatenters through another fluid channel (not shown). This may help to avoidthe possibility of mistakenly illuminating and detecting the samedroplet twice. With or without a wash step, coordinated motions ofvalves as described above may allow an emulsion of sample-containingdroplets to be continuously transferred in and out of any desired numberof detection chambers.

Batch fluorescence detection may be performed without actually stoppingdroplets within the detection chambers of the system. For example, evenif valves 4580, 4582 are not provided or are left open, dropletsentering one of chambers 4568, 4570 may slow sufficiently to allow batchdetection, and the lateral width of the detection chambers may be chosento facilitate this. Alternatively or in addition, various particletracking algorithms may be used to track droplets as they move withinthe detection chambers. Furthermore, a batch detection system may bepartially or completely fluidically decoupled from other portions of amolecular amplification system. For example, a simple array ofdroplet-containing wells or reservoirs (such as a plate array) may beplaced in a fluorescence detection region and imaged as described above.

F. Example 6: Detection Methods

This example describes a method of detecting fluorescence fromsample-containing droplets that have undergone PCR thermocycling; seeFIG. 106.

FIG. 106 is a flowchart depicting the steps of a fluorescence detectionmethod, generally indicated at 4600, which may be performed inconjunction with a PCR system of DNA amplification according to thepresent disclosure. Although various steps of method 4600 are describedbelow and depicted in FIG. 106, the steps need not necessarily all beperformed, and in some cases may be performed in a different order thanthe order shown in FIG. 106.

At step 4602, sample-containing droplets are separated by a desiredaverage distance. This may be accomplished, for example, by various flowfocusing techniques such as those described above (i.e., by flowfocusing the droplets as they are generated), and/or by generatingdroplets at a suitable rate. In cases of batch detection such as in astop-flow system, it may be appropriate for droplets to remain closelyspaced during fluorescence detection, and accordingly a dropletseparation step may not be performed.

At step 4604, the sample-containing droplets are transferred into aradiation intersection region, within which they will be exposed toilluminating radiation chosen to stimulate emission of fluorescenceradiation from one or more fluorescent probes within the droplets, withan intensity that depends in part on whether or not a quenching moietyhas been cleaved from the probe due to polymerase binding of theassociated nucleotide target primer. In the case of continuous flowdetection, the intersection region may be disposed within a fluidchannel such as a capillary tube. In the case of batch detection, theintersection region may be disposed within one or more detectionchambers. In this case, transferring droplets into the intersectionregion may include steps such as opening and closing one or more valvesto allow a continuous flow of droplets into and out of the intersectionregion.

At step 4606, the droplets in the radiation intersection regionencounter and are irradiated with stimulating radiation, which includesat least one wavelength chosen to excite the fluorescent probe(s) knownto be present in the reagents within the droplets. As described above,the illuminating radiation may be produced by a laser, and LED, or anyother suitable radiation source, and may be transferred to theintersection region through free space or through one or more opticalfibers. Furthermore, the radiation may be focused, diverged, split,filtered, and/or otherwise processed before reaching the intersectionregion, to efficiently irradiate the droplets in the most suitablemanner for a particular detector system configuration.

At step 4608, radiation scattered from the droplets in the intersectionregion may be detected by a forward scattering detector. This step willtypically not be performed in a batch detection system, where eachdroplet is approximately stationary or at least relatively slow movingin a detection chamber that serves as the radiation intersection region.However, detecting scattered radiation in a continuous flow detectionsystem may help to correlate simultaneous or subsequent fluorescencedetection with the presence of droplets in the intersection region, andmay allow the volume and target molecule concentration of each dropletto be estimated, as described above. More generally, step 4608 mayinclude performing any measurement to enable an estimation of the volumeof each droplet, such as the amount of radiation scattered from thedroplet, the time of flight of the droplet as it passes through theintersection region, an electrical property of the droplet, or a thermalproperty of the droplet. Method 4600 also may include estimating thevolume of each droplet based on the measurement performed in step 4608.

At step 4610, fluorescence emitted by droplets irradiated in theintersection region is detected by a fluorescence detector. As describedin the preceding examples, the emitted radiation may be transferred tothe fluorescence detector with or without passing through one or moreintermediate optical elements such as lenses, apertures, filters, or thelike. The emitted radiation also may or may not be transferred to thefluorescence detector through one or more optical fibers. In batchdetection applications, the detector and/or the intersection region maybe configured to move in a manner that allows an optical scan of theintersection region by a detector having a smaller field of view thanthe entire intersection region.

At step 4612, detected fluorescence is analyzed to determine whether ornot a particular target nucleotide sequence was present in the droplets.Additional information, including but not limited to an estimate of thenumber or fraction of droplets containing a target molecule, the averageconcentration of target molecules in the droplets, an error margin,and/or a statistical confidence level, also may be extracted from thecollected data.

Using the data collected from each droplet in an analysis may beconditional and may depend, for example, on whether the estimated volumeof the droplet falls within a particular predetermined range. Morespecifically, if the estimated volume of a droplet falls within apredetermined range, then the fluorescence intensity emitted by thatdroplet may be used in a determination of target molecule concentrationin the sample, whereas if the estimated volume of the droplet fallsoutside the predetermined range, then the fluorescence intensity emittedby the droplet may be excluded from a determination of target moleculeconcentration in the sample.

G. Example 8: Additional Embodiments

This example describes additional aspects of sample detection, inaccordance with aspects of the present disclosure, presented withoutlimitation as a series of numbered sentences.

1. A method of detecting target molecule concentration in a sample,comprising (A) generating sample-containing droplets with a dropletgenerator; (B) amplifying target molecules within the droplets; (C)transferring the droplets through an intersection region where thedroplets encounter radiation from a radiation source; (D) estimating thevolume of each droplet based on a measurement performed as the dropletpasses through the intersection region; (E) detecting fluorescenceintensity emitted by each droplet; and (F) for each droplet, if theestimated volume of the droplet falls within a predetermined range thenusing the fluorescence intensity emitted by the droplet in adetermination of target molecule concentration in the sample, and if theestimated volume of the droplet falls outside the predetermined rangethen excluding the fluorescence intensity emitted by the droplet from adetermination of target molecule concentration in the sample.

2. The method of paragraph 1, wherein the measurement is an amount ofradiation scattered from the droplet.

3. The method of paragraph 1, wherein the measurement is time of passageof the droplet through a detector field of view.

4. The method of paragraph 1, wherein the measurement is an electricalproperty of the droplet.

5. The method of paragraph 1, wherein the measurement is a thermalproperty of the droplet.

6. The method of paragraph 1, further comprising separating the dropletsby a desired average distance prior to transferring them through theintersection region.

7. A fluorescence detection method, comprising (A) generatingsample-containing droplets; (B) separating the droplets by a desiredaverage distance; (C) transferring the droplets to a radiationintersection region; (D) exposing the droplets to radiation configuredto stimulate emission of fluorescence radiation from a fluorescent probewithin the droplets; and (E) detecting fluorescence radiation emitted bythe droplets.

8. The method of paragraph 7, wherein separating the droplets includesflow focusing the droplets as they are generated.

9. The method of paragraph 7, further comprising analyzing the detectedfluorescence radiation to determine whether or not each droplet containsa target molecule.

10. A target molecule detection system, comprising (A) a dropletgenerator configured to generate sample-containing droplets; (B) amolecular amplifier configured to replicate target molecules within thedroplets; (C) a radiation source configured to stimulate emission offluorescence radiation from droplets containing target molecules; (D) afluorescence detector configured to detect fluorescence radiationemitted by the droplets; and (E) a first optical fiber configured totransmit stimulating radiation from the radiation source to thedroplets.

11. The system of paragraph 10, wherein the first optical fiber has along axis oriented substantially parallel to a droplet input fluidchannel configured to transport the droplets to an intersection regionwhere the droplets encounter stimulating radiation transmitted by thefirst optical fiber.

12. The system of paragraph 10, wherein the first optical fiber has along axis oriented substantially parallel to a side-facing region of adroplet input fluid channel configured to transport the droplets to anintersection region where the droplets encounter stimulating radiationtransmitted by the first optical fiber, and wherein the side-facingregion is configured to allow substantially only one droplet at a timeto travel parallel to the long axis of the first optical fiber.

13. The system of paragraph 11 or 12, wherein the first optical fiber isfurther configured to transmit fluorescence radiation from the dropletsto the fluorescence detector.

14. The system of paragraph 10, further comprising a second opticalfiber configured to transmit fluorescence radiation from the droplets tothe fluorescence detector.

15. The system of paragraph 14, further comprising a scattering detectorconfigured to detect radiation scattered from the droplets, and a thirdoptical fiber configured to transmit the scattered radiation to thescattering detector.

16. The system of paragraph 10, further comprising (F) a droplet inputfluid channel; and (G) a radiation input fluid channel; wherein thedroplet input fluid channel is configured to transport a fluidcontaining the droplets through an intersection region, the firstoptical fiber is configured to emit radiation from the radiation sourcedirectly into fluid within the radiation input fluid channel, theradiation input fluid channel is configured to transmit radiation fromthe first optical fiber to the intersection region, and the dropletinput fluid channel is fluidically connected to the radiation inputfluid channel.

17. A target molecule detection system, comprising (A) a dropletgenerator configured to generate sample-containing droplets; (B) amolecular amplifier configured to replicate target molecules within thedroplets; (C) a fluid channel configured to transport the dropletsthrough a radiation intersection region; (D) a plurality of radiationsources, each configured to transmit fluorescence stimulating radiationto a different adjacent portion of the intersection region; and (E) atleast one fluorescence detector configured to detect fluorescenceradiation emitted by droplets disposed within the intersection region.

18. The system of paragraph 17, wherein the at least one fluorescencedetector includes a plurality of fluorescence detectors, each configuredto detect fluorescence radiation emitted by droplets within one of thedifferent portions of the intersection region.

19. The system of paragraph 18, wherein each fluorescence detector isconfigured to detect fluorescence radiation at a different wavelength,each wavelength corresponding to at least one variety of targetmolecule.

20. The system of paragraph 19, wherein each radiation source isconfigured to transmit fluorescence stimulating radiation at a differentwavelength.

21. A target molecule detection system, comprising (A) a dropletgenerator configured to generate an emulsion of sample-containingdroplets; (B) a molecular amplifier configured to replicate targetmolecules within the droplets; (C) a droplet input fluid channelconfigured to transfer the emulsion to at least one fluorescencedetection chamber; (D) a radiation source configured to illuminatedroplets within the at least one detection chamber with stimulatingradiation; and (E) a fluorescence detector configured to detectfluorescence radiation emitted by the illuminated droplets.

22. The system of paragraph 21, wherein the at least one detectionchamber is configured to contain substantially only a monolayer ofdroplets.

23. The system of paragraph 21, wherein the at least one detectionchamber includes two detection chambers and a valve mechanism configuredto selectively direct droplets toward one of the two detection chambers.

24. The system of paragraph 23, wherein the valve mechanism isconfigured to allow a substantially continuous flow of emulsion to betransferred from the droplet input fluid channel to the fluorescencedetection chambers.

VII. QUANTIFICATION/ANALYSIS

This Section describes exemplary systems for analyzing reaction dataand, optionally, for using results of the analysis to adjust systemparameters to improve the quality of subsequent data, for example, foruse with droplet-based assay systems. The systems are described, forconvenience, in terms of fluorescence intensity data obtained inconnection with PCR; however, the systems apply more generally todiscrete data obtained in connection with any suitable reaction.Additional pertinent disclosure may be found in the U.S. provisionalpatent applications listed above under Cross-References and incorporatedherein by reference, particularly Ser. No. 61/277,216, filed Sep. 21,2009.

It may be desirable, once a sample-containing emulsion has been created,thermocycled by an enzymatic amplification system such as a PCRthermocycler, and passed through a detection system, to analyze the datagathered by the detection system to extract desired information aboutthe sample. As described previously, the gathered data will typicallyinclude at least a fluorescence intensity level emitted by each detecteddroplet under excitation from a radiation source. The fluorescenceintensity emitted by a given droplet typically will reflect the numberof replicated target nucleic acid molecules in the droplet, and thuswill be a measure of the target molecule concentration in the original,unamplified sample. Fluorescence intensity will be measured by one ormore fluorescence detectors such as a photomultiplier tube or aphotodiode or a digital camera. For example, the fluorescence signalsfrom the detector may be digitized and a peak intensity determined aseach droplet passes within the field of view of the detector. The peakintensity may be determined using a curve fitting technique such as alocal parabolic fit or any other suitable method.

Aside from fluorescence intensities, various other data may be gatheredduring the detection phase. For example, the time of passage of eachdroplet in front of either a fluorescence detector or a forward scatterdetector may be measured. In conjunction with knowledge of the emulsionfluid velocity as it passes through the detection region, and thegeometry of each droplet, this may allow an estimate of each droplet'svolume. Droplet volume also can be estimated by measuring various one ormore other properties of the droplets, such as thermal or electricalconductivity, capacitance, and/or dielectric permittivity, among others.

In any event, it is expected that there will be data, at least includingfluorescence intensity, available for each of a relatively large numberof sample-containing droplets. This will generally include thousands,tens of thousands, hundreds of thousands of droplets, or more.Statistical tools generally may be applicable to analyzing this data.For example, statistical techniques may be applied to determine, with acertain confidence level, whether or not any target molecules werepresent in the unamplified sample. This information may in some cases beextracted simply in the form of a digital (“yes or no”) result, whereasin other cases, it also may be desirable to determine an estimate of theconcentration of target molecules in the sample, i.e., the number oftarget molecules per unit volume.

Because target molecule concentration depends not just on the number oftarget molecules within the emulsion but also on the volume of eachdroplet, determining the target concentration generally also involveseither an explicit or an implicit determination of the volumedistribution of the droplets. In some cases, a droplet volumedistribution may be determined by measuring parameters such as time ofpassage of the droplets in the field of view of a detector, or variousthermal or electrical properties of each droplet, as noted above. Inother cases, the droplet sizes may be assumed to have a certain uniformvalue, for instance based on knowledge of the underlying characteristicsof the droplet generator(s) used in the system. Knowledge of dropletvolumes generally facilitates a determination of the concentration oftarget molecules per unit volume of sample-containing fluid.

Using statistical methods, it is possible to estimate target moleculeconcentration even when the droplet volumes are unknown and no parameteris measured that allows a direct determination of droplet volume. Morespecifically, because the target molecules are assumed to be randomlydistributed within the droplets, the probability of a particular dropletcontaining a certain number of target molecules may be modeled by aPoisson distribution function, with droplet concentration as one of theparameters of the function.

If the droplets are assumed to have a known average size but an unknownsize distribution, the detected fluorescence data, or a quantitycalculated from that data, may be compared to the results predicted byvarious concentration values. The actual concentration value then may beestimated using an error minimization technique such as a least meansquares (LMS) fit.

Even when the droplets are not assumed to be uniform in size, targetconcentration may be estimated in a similar manner. To accomplish this,a particular functional form, such as a Gaussian distribution with aparticular mean and standard deviation, may be assumed for theprobability distribution of droplet volumes. A new Poisson-typedistribution function for the probability of finding a given number oftarget molecules in a droplet then may be calculated, again assuming arandom distribution of target molecules throughout the sample. Anestimate of the target concentration again may be obtained by comparingone or more quantities determined from the actual fluorescence data withthe same quantities predicted by various concentration values, andapplying an error minimization technique as described previously.

Statistical techniques also may be applied to improve the accuracy ofthe data analysis in various ways. For example, statistical analysis offluorescence data may help to determine an appropriate choice of athreshold fluorescence level between negative and positive detection ofa target molecule within a given droplet. Applying this detectionthreshold to the data then may result in a more accurate determinationof target concentration than simply choosing a threshold value a priori.Alternatively, the detection threshold may be left as a variable, andinformation may be extracted from the data across a range of differentthreshold values spanning a portion of (or all of) the range of detectedfluorescence intensities.

Furthermore, the confidence level of the detection thresholdfluorescence level may be increased (or equivalently, the confidenceinterval for a given confidence level may be narrowed) using variousstatistical resampling techniques such as random sampling withreplacement (known in the field of statistics as “bootstrapping”) ofsubsets of the fluorescence data (known as “jackknifing” or “jackknifebootstrapping”). In either case, an improved confidence level in thedetection threshold may be obtained by analyzing the variability of thethreshold level across replacement data sets.

Similarly, statistical methods may be used to provide other forms offeedback that can result in more efficient use of the amplificationsystem and/or more accurate data analysis. For example, an initialdetermination of target molecule concentration in the unamplifiedsample-containing droplets may reveal that the concentration is eithertoo high or too low to be optimal, and this information may be used toadjust various parameters of the system. More specifically, if thetarget concentration is too low (but nonzero), many droplets may containno target molecules at all, resulting in poor statistics and wastedresources in preparing and processing large numbers of “empty” dropletsdespite the fact that some target molecules are present in the sample.On the other hand, if the target concentration is too high, virtuallyall of the droplets will be saturated with target molecules afteramplification, and it will not be possible to determine the targetconcentration of the original sample accurately because there will be nosignificant fluorescence variation among droplets. Either of thesesituations may result in an undesirably large confidence interval forthe determination of target concentration.

Several system parameters may be adjusted in response to a determinationthat the concentration of target molecules in the unamplifiedsample-containing droplets is not optimal for the existing parameters.For example, the sample-containing solution may be diluted orconcentrated prior to droplet generation, to respectively decrease orincrease target concentration. Similarly, the size range of thegenerated droplets may be increased to lower the probability of dropletsbecoming saturated with the target molecule after amplification, ordecreased to increase the likelihood of finding a target molecule (andthe average number of target molecules) in each droplet. In addition,various characteristics of the amplification system, such as thethermocycling temperatures and/or the number of thermocycles, may beincreased in response to a determination that too little amplificationis occurring, or decreased in response to a determination that too muchamplification is occurring.

FIG. 107 is a flowchart depicting a method, generally indicated at 4800,of determining target molecule concentration in a plurality ofsample-containing droplets. As described below, method 4800 includes afeedback mechanism that can be used to adjust one or more parameters ofdroplet generation in response to an undesirably low confidencecondition in the concentration value.

At step 4802, a confidence condition is chosen. This condition caninclude, for example, a desired confidence level and/or an associatedconfidence interval.

At step 4804, sample-containing droplets are generated. Various methodsand apparatus for generating such droplets are described elsewhereherein, for example, in Sections III and IV.

At step 4806, target molecules within the droplets are amplified by PCRor some other enzymatic amplification technique. Methods and apparatusfor amplifying target nucleotide sequences are described elsewhereherein, for example, in Section V.

At step 4808, data such as fluorescence intensity, time of passage, oneor more thermal properties, and/or one or more electrical properties,are collected from the droplets. Methods and apparatus for detectingproperties of sample-containing droplets are described elsewhere herein,for example, in Section VI.

At step 4810, a measure of target molecule concentration (i.e., thenumber of target molecules per unit volume) in the unamplified sample isestimated from the collected data. The estimated measure may include thefraction of droplets containing one or more target molecules, and/or anestimate of the actual concentration.

At step 4812, a confidence condition for the measure estimated in step4810 is determined. Typically, this will include a confidence leveland/or an associated confidence interval, which can be compared to thedesired confidence condition received at step 4802.

At step 4814, the determined confidence condition is compared with thedesired confidence condition, and at step 4816, a determination is madeas to whether the desired confidence condition has been attained.

At step 4818, if the desired confidence condition was attained by theestimated measure of step 4810, then the measure is accepted.

At step 4820, if the desired confidence condition was not attained bythe estimated measure of step 4810, then a determination is made as towhether a suitable droplet generation parameter adjustment is available.Suitable adjustments may include adjusting the number of dropletsgenerated (i.e., generating more droplets), changing the samplechemistry, diluting or concentrating the sample prior to dropletgeneration, generating droplets of different sizes, adjustingthermocycling temperatures, and/or adjusting the number of thermocyclesapplied to the droplets, among others.

At step 4822, if step 4820 determines that a suitable droplet generationparameter adjustment is available, then one or more droplet generationparameter adjustments is made, and the process returns to step 4804 togenerate additional droplets using the adjusted parameter(s). Theparameter adjustment may in some cases be simply to generate moredroplets to improve statistical confidence, without changing any otherparameter of the system. In other cases, a sufficient number ofappropriate droplets already may have been created, and the parameteradjustment may relate entirely to the thermocycler. In that case, step4804 need not be performed again, but rather the method may proceeddirectly from step 4822 back to step 4806. In any event, the method thenproceeds cyclically as FIG. 107 depicts, until either the desiredconfidence condition is met or until no further parameter adjustmentscan be made. In some cases, it may not be possible to further adjust anydroplet generation parameters even if the desired confidence conditionhas not been attained, for example, because of chemical, physical,and/or technological limitations. In this case, i.e., if step 4820determines that a suitable droplet generation parameter adjustment isnot available, then the measure of step 4810 is again accepted at step4818, although it was not possible to meet the desired confidencecondition.

Given a set of droplet fluorescence data, there are various techniquesthat can be used to estimate concentration measures and a confidencecondition such as confidence level and confidence interval. Thefollowing examples describe several specific statistical techniques thatmay be applied to the data to extract useful information to a desireddegree of accuracy under various circumstances.

A. Example 1

This example describes techniques for estimating the concentration perdroplet (average number of target molecules per droplet) with the use ofsome pre-determined calibration or knowledge on the data set, nominallya characteristic such as a fluorescence threshold that may be used todistinguish target-containing droplets from empty droplets, and thestatistical characterization of the confidence of this determination.This example assumes that a collection of values representing thefluorescence intensity for each droplet is available. The techniquesdescribed in this example can be applied to peak fluorescence data(i.e., the maximum fluorescence intensity emitted by a dropletcontaining a particular number of target molecules), but are not limitedto this type of data. The described techniques may be generalized to anymeasurements that could be used to distinguish target-containingdroplets from empty droplets.

If C is the target concentration of a sample (number of target moleculesper unit volume), V_(d) is the volume of a droplet (assumed constant inthis example), and λ=CV_(d) is the average number of copies per droplet,the probability that a given droplet will contain k target molecules isgiven by the Poisson distribution:

$\begin{matrix}{{P\left( {k;\lambda} \right)} = \frac{\lambda^{k}{{Exp}\left( {- \lambda} \right)}}{k!}} & (1)\end{matrix}$

If, for example, there is an average of 3 copies of target nucleic acidper droplet, Poisson's distribution would indicate that an expected 5.0%of droplets would have zero copies, 14.9% would have one copy, 22.4%would have 2 copies, 22.4% would have 3 copies, 16.8% would have 4copies, and so on. It can be reasonably assumed that a droplet willreact if there is one or more target nucleic acid molecules in thevolume. In total, 95% of the droplets should be positive, with 5%negative. Because the different numbers of initial copies per dropletcan, in general, be distinguished after amplification, a generaldescription of the analysis taking this into account can provideimproved accuracy in calculating concentration.

FIG. 108 displays a sample data set where the number of detecteddroplets is plotted as a histogram versus a measure of fluorescenceintensity. The data indicates a peak in droplet counts at an amplitudeof just less than 300, and several peaks of different intensitypositives from about 500 to 700. The different intensity of thepositives is the result of different initial concentrations of targetmolecules. The peak at about 500 had one initial copy, the peak at about600 had two initial copies, and so on until the peaks becomeindistinguishable.

We can define an initial number of copies K after which there is nodifference in detection probability. We can now define a variable, X,describing the probability that a given fluorescence measurement will bedefined as a positive detection (X=1). As equation (2) below indicates,this is defined to be the sum of the probabilities of a dropletcontaining any fluorescently distinguishable positive (first term righthand side) plus the fluorescently saturated positives (second term righthand side), plus the negatives that are incorrectly identified aspositives (third term right hand side):

$\begin{matrix}{{P_{measurement}\left( {X = 1} \right)} = {{\sum\limits_{1 \leqq i < K}^{\;}\; {P_{d_{i}}{P\left( {k = i} \right)}}} + {P_{d_{K}}{P\left( {k \geqq K} \right)}} + {P_{fa}{P\left( {k = 0} \right)}}}} & (2)\end{matrix}$

This can also be written in terms of λ by substituting equation (1) forthe Poisson probabilities:

$\begin{matrix}{{P_{measurement}\left( {X = 1} \right)} = {{\sum\limits_{1 \leqq i < K}^{\;}\; {P_{d_{i}}\frac{\lambda^{t}{{Exp}\left( {- \lambda} \right)}}{t!}}} + {P_{d_{K}}\left\{ {1 - {\sum\limits_{0 \leqq i < K}\frac{\lambda^{t}{{Exp}\left( {- \lambda} \right)}}{t!}}} \right\}} + {P_{fa}{{Exp}\left( {- \lambda} \right)}}}} & (3)\end{matrix}$

The probability that a given measurement will be defined as a negative(X=0) can also be defined as:

P _(measurement)(X=0)=1−P _(measurement)(X=1)  (4)

The equations above are simplified for an apparatus where K=1, i.e.,where one or more target copies per droplet fall within the samefluorescence peak or the separation between positive and negatives is soclear that P_(fa) can be neglected. In some cases, however, there may besignificant overlap between fluorescence peaks of the negative dropletsand the positive droplets, so that P_(fa) is not negligible. Thisexample applies in either case.

The mean of the variable X is the sum of the product of the realizationsand the probabilities:

M _(measurement)=1(P(X=1))+0(P(X=0))=P(X=1)  (7)

or

$\begin{matrix}{M_{measurement} = {{\sum\limits_{1 \leqq i < K}^{\;}\; {P_{d_{i}}\frac{\lambda^{t}{{Exp}\left( {- \lambda} \right)}}{t!}}} + {P_{d_{K}}\left\{ {1 - {\sum\limits_{0 \leqq i < K}\frac{\lambda^{t}{{Exp}\left( {- \lambda} \right)}}{t!}}} \right\}} + {P_{fa}{{Exp}\left( {- \lambda} \right)}}}} & (6)\end{matrix}$

and its standard deviation is given by

$\begin{matrix}{E_{measurement} = \sqrt{\begin{matrix}{{{P_{measurement}\left( {X = 1} \right)}\left( {1 - M_{{measurement}^{\;}}} \right)^{2}} +} \\{{P_{measurement}\left( {X = 0} \right)}M_{measurement}^{2}}\end{matrix}}} & (7)\end{matrix}$

Because the definition of X is such that a negative droplet correspondsto X=0 and a positive droplet corresponds to X=1, the mean of X is alsothe fraction of positive droplets:

$\begin{matrix}{M_{measurement} = \frac{N_{+}}{N}} & (8)\end{matrix}$

Equation 6 and 7 can then be rewritten:

$\begin{matrix}{\frac{N_{+}}{N} = {{\sum\limits_{1 \leq i < K}{P_{d_{i}}\frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}}} + {P_{d_{K}}\left\{ {1 - {\sum\limits_{0 \leq i < K}\frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}}} \right\}} + {P_{fa}{{Exp}\left( {- \lambda} \right)}}}} & (9) \\{\mspace{79mu} {and}} & \; \\{\mspace{79mu} {E_{measurement} = \sqrt{\left( {1 - \frac{N_{+}}{N}} \right)\frac{N_{+}}{N}}}} & (10)\end{matrix}$

Because of their high degree of non-linearity, equations (9) and (10)cannot be readily used to find λ without prior knowledge of theprobabilities P_(di) and P_(fa). A special case occurs when all dropletsare detected (P_(di)=1), only one fluorescent state is distinguishable(K=1), and the positive and negative peaks are easily discernible sothat the probability of a false detection is negligible (P_(fa)=0). Inthis case, equation (9) can be solved for λ:

$\begin{matrix}{\lambda = {\ln \left( {1 + \frac{N_{+}}{N_{-}}} \right)}} & (11)\end{matrix}$

B. Example 2

This example describes extension of the previous example to situationswhere the simplifying assumptions P_(di)=1, K=1, and P_(fa)=0 are notmade. It allows processing the data without the use of somepre-determined calibration or knowledge on the data set. This examplerelies on a least mean squares (LMS) or similar fit of the data to thegeneral theory as outlined by equation (9). We define F as a functiondescribing the difference between the theoretical ratio of droplets (seeequation (9) above) and the measured equivalent:

$\begin{matrix}{F = {{\sum\limits_{1 \leq i < K}{P_{d_{i}}\frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}}} + {P_{d_{K}}\left\{ {1 - {\sum\limits_{0 \leq i < K}\frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}}} \right\}} + {P_{fa}{{Exp}\left( {- \lambda} \right)}} - \frac{N_{+}}{N}}} & (12)\end{matrix}$

This difference should be equal to zero if the proper probabilities andλ can be found. F is, in general, a function of the threshold value setto distinguish positives from negatives, and the distribution offluorescence signals from a set of droplets with the same initial numberof target copies, each of which under basic assumptions can be describedby Gaussian distribution, although other distributions are possible andare conceptually simple extensions of the described method. Morespecifically, due to droplet size variation, PCR efficiency, flow ratevariability through the detector, electrical noise and other such randomfactors, for each number i of initial target molecules in equation (12),there will be a distribution of fluorescence values characterized by amean value, M_(i) and standard deviation σ_(i):

$\begin{matrix}{{P_{i}(t)} = {\frac{1}{\sigma_{i}\sqrt{2\pi}}{{Exp}\left( {- \frac{\left\{ {t - M_{i}} \right\}^{2}}{2\sigma_{i}^{2}}} \right)}}} & (13)\end{matrix}$

The droplets detected as positive from these distributions would bedependent on the chosen threshold:

$\begin{matrix}{P_{d_{i}} = {\int_{Threshold}^{\infty}{\left\lbrack {\frac{1}{\sigma_{i}\sqrt{2\pi}}{{Exp}\left( {- \frac{\left\{ {t - M_{i}} \right\}^{2}}{2\sigma_{i}^{2}}} \right)}} \right\rbrack \ {t}}}} & (14)\end{matrix}$

The function F then becomes:

$\begin{matrix}\begin{matrix}{F\left( {{Threshold},\lambda,M_{i},\sigma_{i}} \right)} \\{{- {\sum\limits_{1 \leq i < K}\left\{ {\int_{Threshold}^{\infty}\left\lbrack {\frac{1}{\sigma_{i}\sqrt{2\pi}}{Exp}} \right.} \right.}}} \\{{\left. {\left. \left( {- \frac{\left\{ {t - M_{i}} \right\}^{2}}{2\sigma_{i}^{2}}} \right) \right\rbrack \ {t}} \right\} \frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}} +} \\{\left\{ {\int_{Threshold}^{\infty}\left\lbrack {\frac{1}{\sigma_{K}\sqrt{2\pi}}{Exp}} \right.} \right.} \\{{\left. {\left. \left( {- \frac{\left\{ {t - M_{K}} \right\}^{2}}{2\sigma_{K}^{2}}} \right) \right\rbrack \ {t}} \right\} \left\{ {1 - {\sum\limits_{0 \leq i < K}\frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}}} \right\}} +} \\{{\left\{ {\int_{Threshold}^{\infty}{\left\lbrack {\frac{1}{\sigma_{0}\sqrt{2\pi}}{{Exp}\left( {- \frac{\left\{ {t - M_{0}} \right\}^{2}}{2\sigma_{0}^{2}}} \right)}} \right\rbrack \ {t}}} \right\} {Exp}}} \\{{\left( {- \lambda} \right) - \frac{N_{+}}{N}}}\end{matrix} & (15)\end{matrix}$

Equation (15) is a general example that applies to a Gaussiandistribution of droplet fluorescence including multiple states ofdetectable positives. A least mean squares fit of equation (15) to aparticular data set may be found through iterative numerical methods,resulting in best fit estimates of λ, M_(i), and σ_(i) for all possiblethreshold settings. The same technique may be applied to any otherwell-defined distribution of target molecules. For example, theconfiguration may be assumed to follow a distribution that takes intoaccount the number of PCR cycles and/or the PCR efficiency.

FIG. 109 shows both the same fluorescence data shown in FIG. 108, againdisplayed as a histogram of the number of droplets detected versus ameasure of fluorescence intensity, and the fluorescence distributionrecreated numerically from equation (15) with several values of K. AsFIG. 109 indicates, the numerically determined function recreates theactual data well, indicating an accurate determination of λ, M_(i), andσ_(i). To determine the numerically optimal fit order, the least meansquare residual between the measured fluorescence data and thenumerically recreated function may be calculated for each fit order, andthe fit order corresponding to the lowest residual may be adopted. Forexample, FIG. 110 is a histogram showing the least mean square residualfor fit orders two through seven obtained with equation (15), showingthat the numerical method becomes increasingly accurate at least up toseven fit orders.

FIG. 111 is a flowchart depicting a method, generally indicated at 4900,for numerically estimating target molecule concentration in a samplebased on aspects of this example. At step 4902, sample-containingdroplets are generated. Various methods and apparatus for generatingsuch droplets are described elsewhere herein, for example, in SectionsIII and IV. At step 4904, target molecules within the droplets areamplified by PCR or some other enzymatic amplification technique.Methods and apparatus for amplifying target nucleotide sequences aredescribed elsewhere herein, for example, in Sections V. At step 4906,data such as fluorescence intensity, time of passage, one or morethermal properties, and/or one or more electrical properties, arecollected from the droplets. Methods and apparatus for detectingproperties of sample-containing droplets are described elsewhere herein,for example, in Section V.

The remaining steps of method 4900 are generally computationallyintensive, and accordingly are typically performed with the aid of adigital processor programmed with suitable instructions. At step 4908, ameasured fraction of droplets containing one or more target molecules isdetermined from the data collected at step 4906. As described above,this fraction will generally be a function of the threshold fluorescencevalue chosen to distinguish a positive (target-containing) droplet froma negative droplet. At step 4910, a theoretical value of the fraction ofdroplets containing one or more target molecules is determined as afunction of target molecule concentration in the original, unamplifiedsample. This theoretical value will, like the value determined from thedata, generally also be a function of the chosen detection threshold. Asuitable theoretical value is provided, for example, by the integralterms of equation (15) above. At step 4912, the target concentration isestimated by minimizing a measure of the difference between thetheoretical fraction determined in step 4910 and the fraction determinedin step 4908 from the collected data. More generally, this step may beperformed by comparing the measured fraction to the theoretical fractionin some manner.

C. Example 3

This example describes methods that may be used to estimate theconfidence interval in an estimated value of target concentration thathas been obtained, for example, using the methods of Examples 1 and 2described above. The confidence interval cannot be directly estimatedwhen a non-linear least mean square is used (as in Example 2). Thebootstrap method, on the other hand, can provide some idea of the errorof the estimation. The principle is based on estimating a plurality ofvalues of target molecule concentration, where each value is estimatedbased on a subset of the collected fluorescence intensity values, andthen determining a mean value and a standard deviation of the estimatedconcentration from the plurality of estimated concentration values. Thesubsets of the samples (here the droplet intensities) are chosenrandomly (Monte Carlo). The standard deviation and mean can then providean estimated concentration as well as a confidence interval defined fromthe standard deviation (if the assumption is that the estimation followsa Gaussian distribution) or directly from the actual results.

One particular type of bootstrap method, which is sometimes referred toas a form of the jackknife bootstrap method, uses data subsets eachchosen to include the total number of data points minus 1. Thismaximizes the statistics available for the estimation while allowing upto the total number of point subsets. This works particularly well for alarge data set. In the case of droplet-based detection, the number ofmeasurements is expected to be on the order of thousands or more, so thejackknife bootstrap technique may be particularly appropriate. In thepresent application of the jackknife bootstrap, this means that eachsubset includes all but one of the collected fluorescence intensityvalues.

The confidence interval obtained using the jackknife bootstrap methodmay be characterized by its dependence on the following factors:

-   -   the number of droplet intensities used in the analysis;    -   the number of data subsets used (the upper limit is the total        number of intensities);    -   the number of threshold values used for the fit; and    -   the fit order.        Numerical studies using sample droplet fluorescence data suggest        the following conclusions regarding these factors:    -   the more droplets, the smaller the confidence interval, with the        confidence interval decreasing approximately as the inverse        square root of the number of droplets;    -   100 jackknife data subsets is typically sufficient to find the        smallest confidence interval for a given set of other        parameters;    -   using a number of different fluorescence thresholds greater than        or equal to approximately a factor of 3 times the number of        unknowns (which equals 2 times the fit order plus one) is        typically sufficient to find the smallest confidence interval        for a given set of other parameters; and    -   the fit order providing the lowest least mean square residuals        should be used.

D. Example 4

This example describes how the methods of the previous examples may beextended to situations in which droplet size is not uniform, but rathervaries among the droplets according to a Gaussian distribution function.The application of equation (15) above relies on the assumption of aconstant droplet volume to calculate the initial concentration, C, fromthe calculated value of λ and the assumed droplet volume V:

λ=CV  (16)

If the droplet volume varies significantly, the same principles can beapplied to solving for the concentration for a given droplet sizedistribution. Equation (2) can be restated as a function of volume:

$\begin{matrix}{{P_{measurement}\left( {{X = 1},V} \right)} = {{{P(V)}{\sum\limits_{1 \leq i < K}{P_{d_{i}}{P\left( {{k = i},V} \right)}}}} + {P_{d_{K}}{P\left( {{k \geq K},V} \right)}} + {P_{fa}{P\left( {{k = 0},V} \right)}}}} & (17)\end{matrix}$

For a Gaussian distribution of droplet volumes with mean M_(V) andstandard deviation σ_(V), equation (15) can be placed into the moregeneral form:

$\begin{matrix}{F\left( {{Threshold},\lambda,M_{v},\sigma_{v},M_{i},\sigma_{i}} \right)} & (18) \\\begin{matrix}{= \left\{ {\int_{0}^{\infty}{\left\lfloor {\frac{1}{\sigma_{v}\sqrt{2\pi}}{{Exp}\left( {- \frac{\left\{ {V - M_{v}} \right\}^{2}}{2\sigma_{v}^{2}}} \right)}} \right\rfloor \ \left( {\sum\limits_{1 \leq i < K}\left\{ {\int_{Threshold}^{\infty}\left\lfloor {\frac{1}{\sigma_{i}\sqrt{2\pi}}{Exp}} \right.} \right.} \right.}} \right.} \\{\left. {\left. {\left. \left( {- \frac{\left\{ {t - M_{i}} \right\}^{2}}{2\sigma_{i}^{2}}} \right) \right\rfloor {t}} \right\} \frac{({CV})^{i}{{Exp}\left( {- {CV}} \right)}}{i!}} \right\} +} \\{{\left\{ {\int_{Threshold}^{\infty}{\left\lbrack {\frac{1}{\sigma_{K}\sqrt{2\pi}}{{Exp}\left( {- \frac{\left\{ {t - M_{K}} \right\}^{2}}{2\sigma_{K}^{2}}} \right)}} \right\rbrack \ {t}}} \right\} \left\{ {1 -} \right.}} \\{\left. {\sum\limits_{0 \leq i < K}\frac{({CV})^{i}{{Exp}\left( {- {CV}} \right)}}{i!}} \right\} +} \\{{\left. {\left\{ {\int_{Threshold}^{\infty}{\left\lceil {\frac{1}{\sigma_{0}\sqrt{2\pi}}{{Exp}\left( {- \frac{\left\{ {t - M_{0}} \right\}^{2}}{2\sigma_{0}^{2}}} \right)}} \right\rceil {t}}} \right\} {{Exp}\left( {- {CV}} \right)}} \right){V}} - \frac{N_{+}}{N}}\end{matrix} & \;\end{matrix}$

Equation (18) can be solved with the same basic principles of least meansquares as equation (15). A knowledge of the mean droplet volume byalternate measurements as described previously, as well as a knowledgeof the standard deviation, will help the least mean square process toconverge to a stable solution. However, the least mean square processcan also be tried without that knowledge, in which case the mean andstandard deviation of the droplet volume will be additional unknownvariables. Additionally, theoretical studies have shown that a standarddeviation of less than 7% of the mean value has a negligible effect onthe results. Therefore, extension of equation (15) to the more generalcase of equation (18) may not be needed for large required confidenceintervals

For the special case where all droplets are detected, P_(di)=1, only onefluorescence state is distinguishable, K=1, and the positive andnegative peaks are easily discernible so that the probability of a falsedetection is negligible, P_(fa)=0, equation (17) will give

M _(measurement)=∫₀ ^(∞) P(V)(1−Exp(−CV))dV  (19)

and the standard deviation becomes:

$\begin{matrix}{E_{meas} = \sqrt{\begin{matrix}{{\int_{0}^{\infty}{{P(V)}\left( {1 - {{Exp}\left( {- {CV}} \right)}} \right)\left( {1 - M_{meas}} \right)^{2}\ {V}}} +} \\{\int_{0}^{\infty}{{P(V)}{{Exp}\left( {- {CV}} \right)}\left( M_{meas} \right)^{2}\ {V}}}\end{matrix}}} & (20)\end{matrix}$

In general, for any known or measured droplet volume distribution P(V),the mean and standard deviation can be calculated.

E. Example 5

This example describes various alternative methods of estimating dropletconcentration, assuming uniform droplet volume and perfect detectability(i.e., all positive droplets detected, and no false detections). Underthese assumptions, an analysis can be performed on the volume spacingbetween positives in the data. It is straightforward to derive theprobability of detecting n negative droplets (i.e., droplets containingno target molecules) before detecting a positive. Applying the Poissondistribution of equation (1), the probability of a droplet containing notarget molecules is:

P(0;λ)=Exp(−λ)=N ⁻ /N  (21)

Therefore, the probability of n consecutive droplets containing notarget molecules is:

[P(0;λ)]^(n)=[Exp(−λ)]^(n)=Exp(−nλ)  (22)

Furthermore, the probability of a droplet containing one or more targetmolecules is:

Σ_(k=1) ^(K=∞) P(k;λ)=1−Exp(−λ)=N ₊ /N  (23)

Thus, the theoretical probability distribution of detecting nconsecutive droplets containing no target molecules before detecting adroplet containing at least one target molecule is:

[P(n;λ)]=[P(0;λ)]^(n)Σ_(k=1) ^(K=∞) P(k;λ)=(1−e ^(−λ))e ^(−nλ)  (24)

Accordingly, the target molecule concentration may be estimated bycomparing the measured probability distribution to this theoreticalprobability distribution. For example, taking the natural log of bothsides,

ln [P(n;λ)]=ln(1−e ^(−λ))−nλ  (25)

Accordingly, a plot of ln [P(n;λ)] versus n will be a line with slope −λand y-intercept ln [1−e^(−λ)], and P(n;λ) as determined from the datamay be used to generate different estimate of λ.

Using equation (24), a related estimator for λ may be derived using amaximum likelihood analysis. Specifically, the value of λ that maximizesthe probability of a spacing of n droplets before detecting a positivedroplet will correspond to the average spacing value. This value may befound by setting the derivative of P with respect to λ equal to zero:

$\begin{matrix}{0 = {\frac{\partial P}{\partial\lambda} - {{n}^{- {n\lambda}}\left( {1 - ^{- \lambda}} \right)} + {{^{- {n\lambda}}\left( ^{- \lambda} \right)}{n = {^{- \lambda}\left( {1 + n} \right)}}{\lambda = {\lambda_{MLE} = {\ln \left( {1 + \frac{1}{\langle n\rangle}} \right)}}}}}} & (26)\end{matrix}$

where <n> is the average value of the spacing calculated from theobserved data, i.e., the average number of droplets containing no targetmolecules before detecting a droplet containing at least one targetmolecule.

F. Example 6

This example describes how the confidence interval for the fraction ofpositive detections can be determined analytically for the case K=1using the central limit theorem. Recall from above that the mean valueof the positive detection ratio may be expressed as:

$\begin{matrix}{M_{measurement} = {{\sum\limits_{1 \leq i < K}{P_{d_{i}}\frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}}} + {P_{d_{K}}\left\{ {1 - {\sum\limits_{0 \leq i < K}\frac{\lambda^{i}{{Exp}\left( {- \lambda} \right)}}{i!}}} \right\}} + {P_{fa}{{Exp}\left( {- \lambda} \right)}}}} & (6)\end{matrix}$

and its standard deviation is given by

$\begin{matrix}{E_{measurement} = \sqrt{\begin{matrix}{{{P_{measurement}\left( {X = 1} \right)}\left( {1 - M_{measurement}} \right)^{2}} +} \\{{P_{measurement}\left( {X = 0} \right)}{M_{measurement}}^{2}}\end{matrix}}} & (7)\end{matrix}$

where the positive detection ratio is measured by the ratio ofpositively detected droplets to the total number of measurements:

$\begin{matrix}{M_{measurement} = \frac{N_{+}}{N}} & (8)\end{matrix}$

The central limit theorem then states that the standard deviation of Nmeasurements is given by E_(measurement)/√N. Therefore, with 95%confidence (2 standard deviations), we have:

$\begin{matrix}{{\frac{N_{+}}{N} \pm {2\frac{E_{measurement}}{\sqrt{N}}}} = {{\sum\limits_{1 \leq i < K}{P_{d_{i}}\frac{\lambda^{i}{\exp \left( {- \lambda} \right)}}{i!}}} + {P_{d_{k}}\left( {1 - {\sum\limits_{0 \leq i < K}\frac{\lambda^{i}{\exp \left( {- \lambda} \right)}}{i!}}} \right)} + {P_{fa}{\exp \left( {- \lambda} \right)}}}} & (27) \\{\mspace{79mu} {with}} & \; \\{\mspace{79mu} {E_{measurement} = \sqrt{\left( {1 - \frac{N_{+}}{N}} \right)\left( \frac{N_{+}}{N} \right)}}} & (28)\end{matrix}$

For simple cases (here shown with K=1, P_(di)=P_(dk)=1, and P_(fa)=0), λcan be derived by inverting the previous equation and the result with aconfidence interval at 95% confidence (2 standard deviations) can beexpressed as:

$\begin{matrix}{{{{\frac{N_{+}}{N} - {2\frac{E_{measurement}}{\sqrt{N}}}} \leq {1 - {\sum\limits_{0 \leq i < K}\frac{\lambda^{i}{\exp \left( {- \lambda} \right)}}{i!}}} \leq {\frac{N_{+}}{N} + {2\frac{E_{measurement}}{\sqrt{N}}}}}\mspace{79mu} {{\frac{N_{+}}{N} - {2\frac{E_{measurement}}{\sqrt{N}}}} \leq {1 - {\exp \left( {- \lambda} \right)}} \leq {\frac{N_{+}}{N} + {2\frac{E_{measurement}}{\sqrt{N}}}}}\mspace{79mu} {1 - \frac{N_{+}}{N} - {2\frac{E_{measurement}}{\sqrt{N}}}}} \leq {\exp \left( {- \lambda} \right)} \leq {1 - \frac{N_{+}}{N} + {2\frac{E_{measurement}}{\sqrt{N}}} - {\ln\left( {1 - \frac{N_{+}}{N} + {2\frac{E_{measurement}}{\sqrt{N}}}} \right)}} \leq \lambda \leq {{- {\ln\left( {1 - \frac{N_{+}}{N} - {2\frac{E_{measurement}}{\sqrt{N}}}} \right)}} - {\ln\left( {1 - \frac{N_{+}}{N} + {2\frac{\sqrt{\left( {1 - \frac{N_{+}}{N}} \right)\left( \frac{N_{+}}{N} \right)}}{\sqrt{N}}}} \right)}} \leq \lambda \leq {- {\ln\left( {1 - \frac{N_{+}}{N} - {2\frac{\sqrt{\left( {1 - \frac{N_{+}}{N}} \right)\left( \frac{N_{+}}{N} \right)}}{\sqrt{N}}}} \right)}}} & (29)\end{matrix}$

G. Example 7

This example describes additional aspects of systems for analyzing dataand improving data collection, in accordance with aspects of the presentdisclosure, presented without limitation as a series of numberedsentences.

1. A method of determining target molecule concentration in a sample toa desired degree of confidence, comprising (A) generatingsample-containing droplets with a droplet generator; (B) amplifyingtarget molecules within the droplets; (C) collecting data from thedroplets including at least values of fluorescence intensity emitted bya plurality of the droplets; (D) estimating target moleculeconcentration in the sample based on the collected data; (E) comparing aconfidence condition for the estimated concentration with a desiredconfidence condition; and (F) if the desired confidence condition hasnot been attained, then adjusting at least one droplet generationparameter.

2. The method of paragraph 1, wherein the droplet generation parameteris the number of droplets generated.

3. The method of paragraph 1, wherein the droplet generation parameteris sample chemistry.

4. The method of paragraph 1, wherein the droplet generation parameteris droplet concentration.

5. The method of paragraph 1, wherein the droplet generation parameteris droplet size.

6. The method of paragraph 1, wherein the droplet generation parameteris a thermocycling temperature.

7. The method of paragraph 1, wherein the droplet generation parameteris a number of thermocycles.

8. The method of paragraph 1, wherein the droplet generator is asingle-use droplet generator.

9. The method of paragraph 1, wherein collecting data includes measuringtime of passage of each of the plurality of droplets in a field of viewof a detector.

10. The method of paragraph 1, wherein collecting data includesmeasuring an electrical property of each of the plurality of dropletssufficient to estimate a volume of each droplet.

11. The method of paragraph 1, wherein collecting data includesmeasuring a thermal property of each of the plurality of dropletssufficient to estimate a volume of each droplet.

12. A method of determining target molecule concentration in a sample,comprising (A) generating sample-containing droplets with a dropletgenerator; (B) amplifying target molecules within the droplets; (C)collecting data from the droplets including fluorescence intensityemitted by each of a plurality of the droplets; (D) determining from thecollected data a measured fraction of droplets containing at least onetarget molecule; (E) determining a theoretical fraction of dropletscontaining at least one target molecule, as a function of targetmolecule concentration; and (F) determining the concentration bycomparing the measured fraction to the theoretical fraction.

13. The method of paragraph 12, wherein the theoretical fraction isdetermined by assuming that the target molecules are randomlydistributed in the droplets.

14. The method of paragraph 12, wherein the theoretical fraction isdetermined by assuming that all droplets are detected, only onefluorescent state is distinguishable, and there are no false detections.

15. The method of paragraph 12, wherein the theoretical fraction isdetermined by assuming that the droplet fluorescence intensities followa Gaussian distribution.

16. The method of paragraph 15, wherein comparing the measured fractionto the theoretical fraction includes minimizing a measure of thedifference between the theoretical fraction and the measured fraction.

17. The method of paragraph 15, wherein comparing the measured fractionto the theoretical fraction includes applying a least mean squares fitof the theoretical fraction to the collected data.

18. The method of paragraph 12, wherein the measured fraction and thetheoretical fraction are both functions of a detection thresholdfluorescence value, and wherein determining the concentration includescomparing the measured fraction to the theoretical fraction for aplurality of detection threshold values.

19. The method of paragraph 12, wherein the theoretical fraction isdetermined by assuming that the droplets have a uniform volume.

20. The method of paragraph 12, wherein the theoretical fraction isdetermined by assuming that the droplets have a Gaussian distribution ofvolumes.

21. A method of determining target molecule concentration in a sample,comprising (A) generating sample-containing droplets with a dropletgenerator; (B) amplifying target molecules within the droplets; (C)collecting data from the droplets including a value of fluorescenceintensity emitted by a plurality of the droplets; (D) estimating aplurality of values of target molecule concentration, wherein each valueis estimated based on a subset of the collected fluorescence intensityvalues; and (E) determining a mean value and a standard deviation of theestimated concentration from the plurality of estimated concentrationvalues.

22. The method of paragraph 21, wherein the subsets are chosen randomly.

23. The method of paragraph 22, wherein each subset includes all but oneof the collected fluorescence intensity values.

24. A method of determining target molecule concentration in a sample,comprising (A) generating sample-containing droplets with a dropletgenerator; (B) amplifying target molecules within the droplets; (C)collecting data from the droplets including a value of fluorescenceintensity emitted by a plurality of the droplets; (D) determining fromthe collected data a measured probability distribution of detecting aparticular number of droplets containing no target molecules beforedetecting a droplet containing at least one target molecule; and (E)estimating target molecule concentration by comparing the measuredprobability distribution to a theoretical probability distribution.

25. The method of paragraph 24, wherein determining the measuredprobability distribution includes determining the average number ofdroplets containing no target molecules before detecting a dropletcontaining at least one target molecule, and wherein estimating targetmolecule concentration includes estimating a maximum likelihood value oftarget molecule concentration.

26. A system for determining target molecule concentration in a sample,comprising (A) a droplet generator configured to generatesample-containing droplets; (B) a thermocycler configured to amplifytarget molecules within the droplets; (C) a fluorescence detectorconfigured to collect values of fluorescence intensity emitted by aplurality of the droplets; and (D) a digital processor configured toestimate target molecule concentration in the sample based on thecollected data, compare a confidence condition for the estimatedconcentration with a desired confidence condition, and send a signal toadjust at least one parameter of the droplet generator or thethermocycler if the desired confidence condition has not been attained.

27. A method of analyzing a sample for a nucleic acid target, comprising(A) removing a portion of a sample; (B) creating an assay mixture fromthe portion for amplification of the nucleic acid target, if present;(C) generating a packet of droplets from the assay mixture, the packethaving an at least substantially predetermined number of the droplets;(D) subjecting the packet to conditions for nucleic acid amplification;(E) performing one or more measurements on each of a plurality ofdroplets of the packet; (F) using the one or more measurements todetermine a number of the plurality of droplets in which amplificationof the nucleic acid target occurred; and (G) estimating the totalpresence of molecules of the nucleic acid target in the sample based onthe number of droplets determined.

28. A method of analyzing a sample for a nucleic acid target, comprising(A) generating a first packet of droplets from a sample, each droplethaving a composition capable of amplification of the nucleic acidtarget, if present, in the droplet; (B) subjecting the first packet toconditions for nucleic acid amplification; (C) performing one or moremeasurements on each of a plurality of droplets of the first packet; (D)using the one or more measurements to determine a first fraction ofdroplets in which amplification of the nucleic acid target occurred; (E)determining whether the first fraction satisfies a predefined confidencecondition for estimating the total presence of target in the sample and,if not, repeating the steps of subjecting, performing, and using with asecond packet of droplets having a different average droplet volumeand/or a different concentration of the sample relative to the firstpacket, to determine a second fraction of droplets in whichamplification of the nucleic acid target occurred; and (F) estimatingthe total presence of the nucleic acid target in the sample based on thefirst fraction, the second fraction, or both.

29. A method for quantifying nucleic acid concentration and/orconfidence interval, comprising (A) providing a sample having a givenstarting volume, the sample to be analyzed for the presence of a targetnucleic acid molecule, (B) removing a sub-sample of a predeterminedvolume from the sample; (C) creating an assay mix by performing thesteps of any of combining the sub-sample with any of primers and orprobes for replicating the target nucleic acid molecule, diluting thesubsample, concentrating the sub-sample, and combinations thereof; (D)creating a droplet packet having a pre-determined number of dropletsfrom the assay mix, the droplets having a droplet volume distribution;(E) subjecting the packets to conditions for nucleic acid replication;(F) performing one or more measurements on at least one droplet from thepackets; (G) using the one or more measurements to determine the numberof droplets comprising replicated target nucleic acid molecules andoptionally droplet volume if the volume distribution is unknown; and (H)using the number of droplets comprising replicated target nucleic acidmolecules to estimate any of the number of target nucleic acid moleculesin the sample volume and the confidence interval thereof andcombinations thereof.

30. The method of paragraph 29, wherein droplet volume is measured usingoptical (e.g., scattering, fluorescence) or electrical (e.g.,conductivity, dielectric permittivity) methods.

31. The method of paragraphs 29 or 30, wherein nucleic assay replicationis measured using optical (e.g., luminescence, fluorescence) orelectrical (e.g., conductivity, dielectric permittivity) methods.

32. A method for determining concentration by changing droplet volume,comprising (A) adjusting the sample partition volume and measuring thepercentage of partitions that amplified at least until finding theresponsive range at which point the concentration can be determined, (B)creating a predetermined number of droplets at first volume that has aconcentration range spanning a responsive range, (C) subjecting thedroplets to conditions that support amplification, (D) measuring asignal (physical property) to determine if amplification has occurredand if DNA was present in the droplets prior to amplification, (E)counting droplets and determining the percentage of droplets thatamplified, (F) estimating the concentration range using the percentagedroplets amplified from Poisson curve or derivative thereof, and (G) ifthe estimated concentration is outside the optimal range then performinganalysis using a second droplet volume.

33. A method of determining concentration by diluting or concentratingdroplet solution, by analogy with paragraph 32.

VIII. Controls and Calibrations for Droplet-Based Assays

This Section describes exemplary control and calibration systems,including methods and apparatus, for example, for performingdroplet-based assays, such as tests of nucleic acid amplification, thatare controlled and/or calibrated using signals detected from droplets.Additional pertinent disclosure may be found in the U.S. provisionalpatent applications listed above under Cross-References and incorporatedherein by reference, particularly Ser. No. 61/275,731, filed Sep. 1,2009.

A. INTRODUCTION

Droplet-based tests for amplification generally need to be accurate. Ifinaccurate, these tests can generate erroneous results, that is, falsenegatives and false positives. Each type of erroneous result can havedetrimental consequences. False negatives related to detection of adisease could mean that the disease is not treated early and ispermitted to spread. In contrast, false positives could causeunnecessary alarm, potentially triggering an unnecessary response thatmay be costly and disruptive. To avoid problems associated with falsenegatives and false positives, inaccurate amplification tests must berepeated to improve their reliability, which increases cost and usesmore sample and reagent, each of which may be precious.

FIG. 112 shows a graph 5710 illustrating an exemplary approach for usingfluorescence to measure amplification of a nucleic acid target indroplets formed by partitioning a sample. The graph plots, with respectto time, fluorescence signals that may be detected from a flow streamcontaining the droplets. Each droplet may be detected as a transientchange (e.g., a transient increase) in intensity of the fluorescencesignal, such as a peak or spike 5712 (i.e., a wave) formed by thefluorescence signal.

To improve clarity, the illustrative data shown here and in otherfigures of this Section are presented as serial data detected from aflow stream containing droplets. However, the methods disclosed in thisSection also may be applied to droplet image data, which may becollected from a set of droplets in parallel (e.g., see Sections II andVI). Also, to improve clarity, the illustrative data are presented in asimplified form: each peak has no width and projects from a constantbackground signal 5713 formed by detection of a continuous phasecarrying the droplets. However, a signal peak may have any suitableshape based on, for example, the frequency of detecting signals, theshape of each droplet, the size and geometry of a channel carrying theflow stream, the flow rate, and the like. Moreover, the signal peaks mayhave any suitable temporal distribution, for example, occurring atrelatively constant intervals, as shown here, or at varying intervals. Adroplet signal provided by and/or calculated from the peak (e.g., asignal corresponding to peak height or peak area, among others) may beused to determine whether amplification occurred in the correspondingdroplet, and thus whether the droplet received at least one molecule ofthe nucleic acid target when the sample was partitioned.

Each droplet signal may be compared to a signal threshold 5714, alsotermed a cutoff. This comparison may provide a determination of whethereach droplet signal represents a positive signal (target is present) ora negative signal (target is absent and/or not detected), foramplification in the droplet. For example, droplet signals greater than(and, optionally, equal to) the threshold may be considered asrepresenting positive droplets. Conversely, droplet signals less than(and, optionally, equal to) the threshold may be considered asrepresenting negative droplets. (A positive droplet signal abovethreshold 5714 is indicated at 5716, and a negative droplet signal belowthreshold 5714 is indicated at 5718 in FIG. 112.) Comparison to thethreshold thus may transform each droplet signal to a digital value,such as a binary value (e.g., a “1” for a positive droplet and “0” for anegative droplet). In any event, the fraction of droplets that arepositive can be determined. For a given droplet size, the fraction ofpositive droplets can be used as an input to an algorithm based onPoisson statistics to determine the number of copies (molecules) of thenucleic acid target present in the initial sample volume. In someembodiments, more than one threshold may be used to categorize results(e.g., negative, positive, or inconclusive; or no copies, one copy, twocopies, three or more copies; etc.).

FIG. 113 shows an exemplary histogram 5720 of ranges of droplet signalintensities that may be measured from the flow stream of FIG. 112. Therelative frequency of occurrence of each range is indicated by barheight. The distribution of positive and negative signal intensities maybe larger than the modest difference in signal intensity produced byamplification (a positive droplet) relative to no amplification (anegative droplet). Thus, the distributions of droplet signals frompositive droplets and negative droplets may produce a problematicoverlap between the amplification-positive and amplification-negativedroplet signals, indicated at 5724. Accordingly, as shown in FIG. 112,some amplification-positive droplets may provide relatively weak dropletsignals, such as false-negative signal 5726, that are less thanthreshold 5714, resulting in incorrect identification of these positivedroplets as negative. Conversely, some amplification-negative dropletsmay provide relatively strong droplet signals, such as false-positivenegative signal 5728, that are greater than threshold 5714, resulting inincorrect identification of these negative droplets as positive. Sinceeither type of erroneous result may be costly and harmful, it isdesirable to minimize their occurrence.

There are many factors that can lead to variation in signals detectedfrom droplets. Examples of physical parameters that may affect thesignals may include droplet position when detected (e.g., relative tothe “sensed volume” of the detector), droplet volume and shape, opticalalignment of detection optics (including excitation source, filters, anddetector), detector response, temperature, vibration, and flow rate,among others. Examples of reaction chemistry parameters that may affectthe fluorescence signal include the number of target molecules and/orthe amount of background nucleic acid present in each droplet,amplification efficiency, batch-to-batch variations in reagentconcentrations, and volumetric variability in reagent and sample mixing,among others. Variations in these physical and chemical parameters canincrease the overlap in the distribution of positive and negativedroplet signals, which can complicate data interpretation and affecttest performance (e.g., affect the limit of detection). The variationscan occur within a run and/or between runs, within a test on a targetand/or between tests on different targets, on the same instrument and/ordifferent instruments, with the same operator and/or differentoperators, and so on.

Thus, there is a need for improved accuracy and reliability indroplet-based assays. For example, it would be desirable to havedroplet-based controls for these tests, optionally, droplet-basedcontrols that can be incorporated into test droplets (for performingassays) or incorporated into control droplets (for controlling assays)that can be intermixed with test droplets. Such integrated controls mayhave the benefit of reducing cost by processing control reactions inparallel with test reactions, which may speed the analysis. It alsowould be useful to have one or more controls that can be used in systemcalibration (e.g., to verify hardware, reagent, and/or software (e.g.,algorithm) performance, among others).

B. DEFINITIONS

Technical terms used in this disclosure have the meanings that arecommonly recognized by those skilled in the art. However, the followingterms may have additional meanings, as described below.

Signal—detectable and/or detected energy and/or information. Any of thesignals detected, after detection, may be described as signals and/ordata. For example, detected droplet signals may provide test signals andtest data, control signals or control data, reference signals andreference data, calibration signals and calibration data, transformedsignals and transformed data, or any combination thereof, among others.

Transform—to change one or more values, and/or the number, of signals ofa data set using one or more mathematical and/or logical operations.Transformation of a set of signals may produce a transformed set of thesignals by changing values of one or more of the signals and/or bydeleting/invalidating any suitable subset of the signals. Signaltransformation may include reducing signal variation,deleting/invalidating outlier signals, subtracting a baseline value fromsignals, reducing the frequency of outliers, reducing the overlap ofdistributions of positive and negative droplet signals, modifyingsignals according to a regression line, assigning new values to signalsbased on comparing signal values to a threshold or range, or anycombination thereof, among others.

Run—an operating period during which a set of droplets, generallydroplets of about the same size and including partitions of a sample,are tested.

Oligonucleotide—a nucleic acid of less than about one-hundrednucleotides.

Exogenous—originating externally. For example, a nucleic acid exogenousto a sample is external to the sample as originally isolated. As anotherexample, a nucleic acid exogenous to an organism or cell is not nativeto the organism or cell, such as a nucleic acid introduced into theorganism or cell by infection or transfection.

Endogenous—originating internally, such as present in a sample asoriginally isolated or native to a cell or organism.

C. SUMMARY

The present disclosure provides a system, including methods andapparatus, for performing droplet-based tests of nucleic acidamplification that are controlled and/or calibrated using signalsdetected from droplets.

The present disclosure provides a method of sample analysis. Dropletsmay be obtained. The droplets may be generated on-line or at least asubset of the droplets may be pre-formed off-line. At least a subset orall of the droplets may include a partition of a sample to be tested andmay be capable of amplification of at least one test nucleic acidtarget, if present, in the partition. In some embodiments, the dropletsmay be capable of amplification of a test nucleic acid target and acontrol nucleic acid target. The droplets collectively or each mayinclude a dye, or at least a first dye and a second dye. In someembodiments, the droplets may be of at least two types, such as two ormore types of test droplets, test droplets and calibration droplets, ortest droplets and control droplets, among others. In some embodiments,the two or more types of droplets may be distinguishable based ondistinct temporal positions of the droplet types in a flow stream (ordistinct times of exit from the flow stream, e.g., distinct times atwhich the droplets are collected in one or more detection chambers forimaging), the presence of respective distinct dyes in the droplet types,distinguishable signal intensities of the same dye (or different dyes),or a combination thereof, among others.

Signals, such as fluorescence signals, may be detected from thedroplets. The signals may include test signals, calibration signals,control signals, reference signals, or any combination thereof. In someembodiments, test signals and control signals may indicate respectivelywhether amplification of a test nucleic acid target and a controlnucleic acid target occurred in individual droplets. In someembodiments, detection may include (a) exciting first and second dyeswith a same wavelength of excitation light and (b) detecting emittedlight from the first and second dyes at least substantiallyindependently from one another in respective first and second detectorchannels.

The signals detected may be analyzed to determine a test result relatedto a presence (number, concentration, etc.), if any, of a test nucleicacid target in the sample. In some embodiments, analysis may includetransforming test signals based on reference signals to reduce variationin the test signals. The test signals and the reference signals may bedetected in respective distinct detector channels or in the samedetector channel. In some embodiments, the reference signals may beprovided by a second dye that is not coupled to an amplificationreaction and thus serves as a passive reference. In some embodiments,the reference signals may be provided by control signals detected from acontrol amplification reaction. The control amplification reaction maymeasure amplification of an exogenous or endogenous template. In someembodiments, analysis may include (a) comparing test signals, or atransformed set of the test signals, to a signal threshold to assignindividual droplets as positive or negative for a test nucleic acidtarget, and (b) estimating a number of molecules of the test nucleicacid target in the sample based on the comparison. In some embodiments,analysis may include (a) analyzing control signals to determine acontrol value corresponding to a number and/or fraction of the dropletsthat are amplification-positive for a control nucleic acid target, and(b) interpreting a test result, such as determining its validity, basedon the control value.

The systems disclosed herein may offer improved instrument calibrationand/or substantial improvements in the accuracy and/or reliability ofdroplet-based amplification tests. Exemplary capabilities offered by thepresent disclosure may include any combination of (1)correcting/minimizing variations in the fluorescence signal to increasethe accuracy of droplet PCR results; (2) providing an internal indicatorof whether nucleic acid amplification failed (e.g., PCR inhibition frominterfering components in the sample, incorrect sample and reagentmixing, incorrect thermal cycling, incorrect droplet formation); (3)providing measurement of droplet volumes without having to addadditional hardware components; (4) providing measurement of changes inthe baseline fluorescence signal (i.e., baseline drift); (5) providingcalibration of a droplet detector before and/or during a run; (6)monitoring the performance of quantitative droplet PCR measurements anddata processing algorithms before and/or during a run; (7) verificationof droplet integrity (e.g., absence of coalescence); (8) obtaininginformation on droplet generation and detection frequency (spatially andtemporally) using an in-line detector; (9) measuring variations andcomparing them to predefined tolerances; (10) processing of raw dropletPCR data to correct for variations and increase test accuracy andperformance; (11) incorporating control assays preferably using a singleexcitation source; and/or (12) quantifying more than one species ofgenetic target by amplifying and detecting more than one species ofgenetic target in individual droplets.

D. SYSTEM OVERVIEW

FIG. 114 shows an exemplary system 5740 for performing droplet-basedtests of with the aid of controls and/or calibrators. Other exemplarysystems that may be suitable are described elsewhere herein, such as inSection II, among others. System 5740 may include any combination of asample preparation station 5742, at least one droplet generator 5744, aheating station, such as a thermal cycler 5746, a detection station5748, and a controller 5750 incorporating a data analyzer 5752 and afeedback and control portion 5754, among others.

The system may provide at least one flow stream that carries at leastone sample and reagents from one or more upstream positions and in adownstream direction to detection station 5748. Signals detected fromthe flow stream (or detected with stopped flow, such as by imaging), andparticularly droplet signals, may be communicated to data analyzer 5752.The data analyzer may analyze the signals to determine one or more testresults, control results, calibration results, a quality (e.g.,validity, reliability, confidence interval, etc.) of any of the results,or a combination thereof. Any of the results may be communicated tofeedback and control portion 5754, which may control and/or adjustcontrol of any of sample preparation station 5742, droplet generator5744, thermal cycler 5746, detection station 5748, and data analyzer5752, based on the results determined.

Preparation station 5742 may contain and/or supply at least one sample5756, at least one set of test reagents 5758 (also termed targetreagents), one or more control reagents 5760, one or more calibrationreagents 5762, or any combination thereof. Any of the samples and/orreagents may be stored and/or supplied separately, may be stored and/orsupplied as one or more pre-formed mixtures, and/or may be mixedselectably before they are supplied to a downstream region of the system(e.g., droplet generator 5744, thermal cycler 5746, or detection station5748). Furthermore, any of the samples and/or reagents may travelsequentially from sample preparation station 5742 to droplet generator5744, thermal cycler 5746, and then detection station 5748 for detectionof droplet signals. Alternatively, any of the samples and/or reagentsmay reach the detection station without travel through the dropletgenerator, as indicated at 5764, the thermal cycler, or both, asindicated at 5766. Accordingly, any of the samples and/or reagentsdisclosed herein may be stored and/or supplied in pre-formed droplets.Droplets may, for example, be pre-formed off-line, either locally orremotely. Pre-formed droplets may be intermixed randomly with dropletsformed by droplet generator 5744 before reaching detection station 5748,or distinct types of droplets may be detected as spatially and/ortemporally separated packets of droplets.

Test reagents 5758 may be any reagents used to test for amplification ofone or more targets, such as one or more primary targets, in partitionsof a sample. Primary targets generally comprise any targets that are ofprimary interest in a test. Primary targets may be present at an unknownlevel in a sample, prior to performing tests on the sample. Testreagents 5758 generally include one or more sets of target reagentsconferring specificity for amplification of one or more particularnucleic acid targets to be tested in a sample. Thus, the test reagentsmay include at least one pair (or two or more pairs) of primers capableof priming amplification of at least one (or two or more) nucleic acidtarget(s). The test reagents also may comprise at least one reporter tofacilitate detecting amplification of each test target, a polymerase(e.g., a heat stable polymerase), dNTPs, and/or the like. The testreagents enable detection of test signals from droplets.

Control reagents 5760 are any reagents used to control for test signalvariation (generally, variation other than that produced by differencesin amplification) and/or to interpret results obtained with the testreagents (such as a reliability and/or validity of the results). Thecontrol reagents permit control signals and/or reference signals to bedetected from droplets, either the same or different droplets from thetest signals. Control reagents may be mixed with test reagents prior todroplet formation and/or control droplets containing control reagentsmay be produced separately from the test droplets and introducedindependently of the sample.

The control reagents may provide instrument controls, that is, controlsfor variation introduced by the system (and/or its environment). Thus,instrument controls may control for variation in droplet volume, dropletdetection efficiency, detector drift, and the like. Reference signalsmay be detected from droplets containing control reagents that functionas instrument controls.

The control reagents also or alternatively may provide amplificationcontrols, that is, controls that test for secondary/controlamplification in droplets. The control reagents thus may includereagents used to test for amplification of at least one secondary orcontrol target in droplets. The secondary/control target may be ofsecondary interest in a test, and/or may be present at a known orexpected level in the sample, among others. In any event, the controlreagents may include one or more sets of target reagents conferringspecificity for amplification of one or more control nucleic acidtargets to be tested in droplets. The control reagents may include atleast one pair (or two or more pairs) of primers capable of primingamplification of at least one (or two or more) control nucleic acidtarget(s). The control reagents also may comprise at least one reporterto facilitate detecting amplification of each control target, apolymerase (e.g., a heat stable polymerase), dNTPs, and/or the like, orany suitable combination of these control reagents may be supplied bythe test reagents. Control signals may be detected from control reagentsthat function as amplification controls.

Calibration reagents 5762 are any reagents used to calibrate systemoperation and response. Droplets containing a calibration reagent (i.e.,calibration droplets) may be introduced into a flow stream of thesystem, at any position upstream of the detection station, for thepurpose of calibrating the system (e.g., calibrating flow rates,excitation power, optical alignment, detector voltage, amplifier gain,droplet size, droplet spacing, etc.). Calibration droplets may beintroduced into a flow stream of the system before, during, and/or afterintroduction of test droplets into the flow stream. In some embodiments,the level of a dye within control droplets may be used to calibrateand/or validate detector response, such as by using a pair of dyeconcentrations providing calibration signals that bracket an intendedmeasuring range and/or that are disposed near upper and lower ends ofthe measuring range. For example, droplets of known size and containingone or more known dye concentrations may be prepared off-line andintroduced into the system, and/or may be generated by the system. Insome embodiments, calibration droplets may comprise fluorescentparticles such as quantum dots, polymer beads, etc.

System 5740 may used to perform a method of analyzing one or moresamples. The method may include any suitable combination of the stepsdisclosed herein, performed in any suitable order.

Droplets may be obtained. The droplets may be of one type or two or moretypes. At least a subset, or all, of the droplets may be generated bythe system or may be pre-formed off-line. At least a subset of thedroplets may include test reagents for testing amplification of a testnucleic acid target. At least a subset of the droplets may includecontrol reagents and/or calibration reagents for testing amplificationof a control nucleic acid target. The droplets may contain one or moredyes.

The droplets may be introduced into a flow stream upstream of adetector. All of the droplets may be introduced into the flow stream atthe same position or the droplets, particularly droplets of differenttypes, may be introduced at two or more distinct positions.

The droplets, in the flow stream, may be subjected to conditions thatfacilitate amplification. For example, the droplets may be heated and/ormay be heated and cooled repeatedly (thermally cycled).

Signals may be detected from the droplets. The signals may include testsignals, control signals, reference signals, calibration signals, or anycombination thereof.

The signals may be analyzed. Analysis may include transforming testsignals. Analysis also or alternatively may include comparing testsignals and/or transformed test signals to a signal threshold to assignindividual droplets as being positive or negative for amplification of anucleic acid target. A number and/or fraction of target-positivedroplets may be determined based on results of the comparison. Analysisfurther may include estimating a presence of a nucleic acid target inthe sample. The estimated presence may be no target in the sample.Estimation of the presence may (or may not) be performed using Poissonstatistics.

E. EXEMPLARY INSTRUMENT CONTROLS AND CALIBRATIONS

FIG. 115 shows selected aspects of system 5740 in an exemplaryconfiguration 5780 for detecting amplification of a nucleic acid targetusing a first dye and for controlling for system variation during a testusing a second dye. In FIG. 115 and in other system configurationspresented in succeeding figures of the present disclosure, the terms“droplet generator,” “thermal cycler,” and “detection station” areabbreviated “DG,” “TC,” and “DET.”

Sample preparation station 5742 may supply an amplification mixture todroplet generator 5744. The amplification mixture may incorporate asample 5756, target reagents 5782 (i.e., test reagents 5758) including afirst dye 5784 (dye 1), and a second dye 5786 (dye 2). The second dyeand the target reagents may be mixed with one another beforeintroduction into system 5740 or may be mixed within the system. Targetreagents 5782 may provide primers for amplification of a nucleic acidtarget, and the first dye may enable detection of whether amplificationoccurred. The first and second dyes may be fluorescent dyes that aredistinguishable optically. The second dye may be a passive reference orinstrument control. In other words, the second dye may provide adetectable signal having an intensity that is at least substantiallyindependent of the extent of amplification, if any, of any nucleic acidtarget.

Droplet generator 5744 may form droplets of the amplification mixture.The droplets may travel through thermal cycler 5746, to promoteamplification of the nucleic acid target, if any, in each droplet. Thedroplets then may travel to detection station 5748. Station 5748 maydetect, for each droplet, a test signal from the first dye and areference signal (also termed a control signal) from the second dye.

FIG. 116 shows exemplary target reagents 5782 and a control reagent 5760that may be included in system configuration 5780 of FIG. 115. Thetarget and control reagents may permit detection of test signals in afirst detector channel 5788 (“channel 1”) and detection of referencesignals in a second detector channel 5790 (“channel 2”). The first andsecond channels may represent distinct wavelengths and/or at leastsubstantially nonoverlapping wavelength ranges.

Target reagents may include a reporter, such as a probe 5792, andtarget-specific forward and reverse primers 5794. Probe 5792 may be anenergy transfer probe (e.g., a TAQMAN probe) including a nucleic acid,such as an oligonucleotide 5796, that binds to amplified target, and anenergy transfer pair connected to strand 5796. The energy transfer pairmay, for example, be formed by first dye 5784 and a quencher 5798.

Control reagent 5760 may include second dye 5786. The second dye may (ormay not) be connected to a nucleic acid, such as an oligonucleotide5800. Connection to the oligonucleotide may be covalent and/or through abinding interaction. Connection of the second dye to an oligonucleotideor other water-soluble molecule may improve retention of the second dyein the aqueous phase of a droplet and/or may facilitate distribution ofthe dye throughout the aqueous phase, among others.

FIG. 117 shows a flowchart illustrating of an exemplary approach tocorrecting for system variation using system configuration 5780 (FIG.115), and, optionally, the reagents illustrated in FIG. 116. Testsignals (i.e., target signals) and reference signals may be detectedfrom the same droplets. For example, test signals may be detected in afirst channel and reference signals may be detected in a second channel.Graphs illustrating coincident detection of test signals and referencesignals are shown at 5810, 5812, respectively.

Test signal variation may introduce errors in data processing. Forexample, graph 5810 shows substantial variation in the intensity of thetest signals detected. As a result, some of the test signals may beerroneously classified as positives or negatives. In the presentillustration, two false positives are marked. However, variation of thetest signals may be mirrored by variation of the reference signalsdetected from the same droplets. Accordingly, the test signals may betransformed based on the reference signals, indicated at 5814, tocorrect for variation in the test signals, as shown in a graph 5816,which plots the transformed test signals. The test signals may betransformed by any suitable operation or set of operation involving thereference signals. For example, the test signals may be transformedthrough dividing test signals by reference signals, such as dividingeach test signal by its corresponding reference signal, which may bedescribed as normalizing the test signals. Alternatively, the testsignals may be transformed based on the reference signals by, forexample, baseline subtraction, distance from the regression line, or thelike. A transformation may compensate for variations in the testchannel. This compensation or correction may make the test signals(i.e., negative test signals and/or positive test signals) more uniformin value and/or more Gaussian. The transformation also or alternativelymay reduce the frequency of outliers and/or the overlap of thedistributions of positive and negative signals.

FIG. 118 shows selected aspects of system 5740 in an exemplaryconfiguration 5830 for (a) detecting amplification of a nucleic acidtarget in a set of droplets and (b) system calibration and/or correctionfor system variation in another set of droplets. Configuration 5830 issimilar to configuration 5780 of FIG. 115, except that target reagents5782 and control reagent 5760 are not in the same droplets. Accordingly,the target reagents and the control reagent may be supplied torespective distinct droplet generators of the system, indicated at 5832,may be supplied to the sample droplet generator at different times, orthe control reagent may be supplied in pre-formed droplets that do notpass through the droplet generator, indicated at 5834, 5836. Since thetarget reagents and the control reagent are not in the same droplets inthis configuration, the control reagent may include the same dye as thetarget reagent (i.e., first dye 5784) or may include a distinct dye(such as second dye 5786).

FIG. 119 shows an exemplary graph 5850 of fluorescence signals that maybe detected over time from a flow stream of system configuration 5830(FIG. 118) during system calibration, indicated at 5852, and sampletesting, indicated at 5854. Calibration and sample testing may beperformed without or with mixing of calibration and test droplets.

Calibration and sample testing may be performed serially, without mixingof droplet types, using the same dye (and/or detection of the samewavelength(s)). By keeping calibration and test droplets separate, thedistributions of test and calibration signal intensities may overlap.For example, calibration droplets and test droplets may be separatedtemporally in the flow stream, such that each type of droplet isidentifiable based on its time of arrival at the detection station. Thetime of arrival may be calculated based on the relative time ofintroduction of each droplet type into the flow stream and the velocityof the flow stream. Thus, the calibration and test droplets may not (ormay) be distinguishable based on signal intensity, but may bedistinguishable temporally. In particular, the test and calibrationdroplets may be separated by a temporal (and spatial) gap 5856, whichmay identify a transition between droplet types. The use of temporalgaps also may permit introduction of a set of calibration dropletswithin a set of test droplets (i.e., within a test run), with a gappreceding and following the set of calibration droplets, to provideidentification of each transition to a different droplet type. Stateddifferently, calibration may be performed during sample testing, byinserting calibration droplets into a train of test droplets, such thatthe train of test droplets is divided into two or more discrete groups.

Calibration droplets may include two or more types of droplet, which maybe introduced separately or intermixed. For example, FIG. 119 shows aset of stronger calibration signals 5858 followed by a set of weakercalibration signals 5860 produced by distinct types of calibrationdroplets. Stronger and weaker calibration signals 5858, 5860 maycorrespond generally in intensity to respective positive test signals5862 and negative test signals 5864. In other embodiments, only one typeor three or more types of calibration droplet may be used, and may beconfigured respectively to provide one or three or more intensities ofcalibration signals.

Calibration and sample testing alternatively may be performed withcalibration and test droplets randomly intermixed and thus notdistinguishable temporally. Intermixed calibration and test droplets maybe distinguishable by incorporating distinguishable dyes into therespective droplet types and, optionally, by detection of thedistinguishable dyes at respective distinct wavelengths. Alternatively,or in addition, calibration droplets and test droplets may bedistinguishable according to signal intensity detected at the samewavelength(s) and optionally from the same dye. In particular,calibration droplets may be designed to have one or more signalintensities outside the signal range of test droplets (i.e., the signalrange provided by the collective distribution of signal intensities fromnegative and positive test droplets (e.g., see FIG. 113)). Thus,calibration droplets may be identified based on their calibrationsignals having signal intensities above and/or below the signal range oftest droplets.

FIG. 120 shows a flowchart 5880 of an exemplary approach to correctingfor signal variation during an amplification test using systemconfiguration 5830 of FIG. 118. The approach illustrated in FIG. 120distinguishes types of droplet signals, namely, test droplet signals5882 and reference droplet signals 5884, based on differences in signalintensity detected in the same detector channel, as described above forcalibration droplets. In particular, test droplets may produce a range5886 of signal intensities, and reference signals 5884 may haveintensities below (or above) the range. Accordingly, the distinct typesof droplets may be interspersed randomly in the flow stream.

The reference droplets may be formed with the same amount (or two ormore discrete amounts) of dye. Accordingly, without signal variationgenerated by the system, the reference droplets should produce referencesignals of the same intensity. Variation in reference signal intensitymay be mirrored by corresponding changes in the intensity of testsignals. For example, in graph 5888, the intensity of reference signals5884 and negative test signals 5890 show a gradual increase with respectto time. As a result, test signals from amplification-negative dropletsmay produce false positives 5892.

Variation in test signals 5882 may be reduced by transforming the testsignals, indicated at 5894, based on reference signals 5884, to producenormalized test signals 5896 presented in graph 5898. Transformationmay, for example, be performed by transforming each test signal based onone or more reference signals temporally proximate to the test signal, aweighted average of reference signals temporally proximate to the testsignal, a sliding window of averaged reference signals that overlaps thetest signal, or the like. Transformation before comparing test signalsto a threshold may reduce the incidence of false positives, as shownhere, the incidence of false negatives, or both.

F. EXEMPLARY AMPLIFICATION CONTROLS

FIG. 121 show selected aspects of system 5740 of FIG. 114, with thesystem in an exemplary configuration 5910 for testing amplification ofat least a pair of nucleic acid targets in the same droplets. Systemconfiguration 5910 may form an amplification mixture, which is suppliedto droplet generator 5744. The amplification mixture may incorporate asample 5756, test amplification reagents 5858, control amplificationreagents 5912, and at least one control template 5914. Any combinationof the sample, test reagents, control reagents, and control template maybe mixed with one other before introduction into system 5740, or may bemixed within the system. Test reagents 5758 and control reagents 5912may provide primers for respective amplification of at least one testtarget and at least one control target.

Amplification of the test and control targets may, for example, bedetected via a first dye and a second dye, respectively, which may beincluded in respective first and second reporters (e.g., first andsecond probes). Signals from the first and second dyes may be detectedin distinct (e.g., at least substantially nonoverlapping) first andsecond channels (i.e., a test channel and a control channel) as testsignals and control signals, respectively.

Control template 5914 may comprise exogenous molecules of the controltarget. In contrast, the sample may be tested for a presence ofendogenous molecules of the test target. The control template 5914 maybe present in any suitable amount to provide any suitable average numberof control template molecules per droplet, to generate a desiredfraction of droplets positive for the control template. For example, thenumber of template molecules provided by template 5914 may besubstantially less than an average of one per droplet, such as anaverage of about 0.1, 0.05, 0.02, or 0.01 molecule per droplet.Accordingly, the number/concentration of control template molecules maybe selected such that the frequency of amplification of both test andcontrol targets in the same droplet is low, which may minimizecompetition that may be caused by amplification of both test and controltargets. For example, the control template may be present in no morethan about one in five droplets.

The frequency of amplification of the control target may be determinedby performing an analysis with the system. In some embodiments, thisfrequency may be compared with one or more previously determinedfrequencies of amplification for the control target and/or may becompared with an expected value for the frequency provided by amanufacturer. In any event, a control value may be determined, with thecontrol value corresponding to a number and/or fraction of the dropletsthat are amplification-positive for the control nucleic acid target.

Control signals acquired in the control channel may be used to measureand/or verify the quantitative accuracy of a run and/or the measurementprecision of the system during two or more runs. The control signalsalso or alternatively may be used to interpret a test result, such asthe quality of test data measured from a sample, for example, to verifythe quantitative accuracy of the test data and/or to determine thevalidity and/or reliability of the test data. The test result may beinterpreted based on control value determined. For example, the testresult may be determined as being invalid if the control value is lessthan a threshold value. Furthermore, data acquired from the controlchannel, such as signals from amplification-negative control droplets,may provide reference signals, as described above in relation to FIG.117. In other words, test signals may be transformed using controlsignals that functions as reference signals, to normalize the testsignals.

FIG. 122 shows selected aspects of system 5740 of FIG. 114, with thesystem in another exemplary configuration 5920 for testing amplificationof at least a pair of nucleic acid targets in the same droplets. Systemconfiguration 5920 differs from configuration 5910 of FIG. 121 byincluding a different set of control amplification reagents 5922 (or asecond set of test amplification reagents) and by the absence of anexogenous control template. Control reagents 5922 may amplify a controltarget that is known or expected to be present in sample 5756, and/orthat has a known or expected representation with respect to a bulknucleic acid population present in the sample (e.g., total DNA, totalgenomic DNA, genomic DNA from a particular species of organism, totalRNA, total mRNA, etc.). In contrast, target reagents 5758 may amplify atest target that has an unknown presence in the sample and/or an unknownpresence in with respect to the bulk nucleic acid population. In anyevent, amplification of the control target may be used to determine thequality of test data measured from a sample, such as to verify thequantitative accuracy of the test data and/or to determine thereliability of the test data. Furthermore, an amount of control targetdetermined to be present in the sample may provide a standard againstwhich an amount of test target determined to be present in the samplecan be compared and/or normalized. In some embodiments, a control targetis selected that is rare in the sample, such as a target representing aparticular gene mutation. By selecting a rare control target,amplification of the control target can indicate the limit of detectionof a test target and/or whether amplification of a low-abundance testtarget can occur. In some embodiments, the control target may bereplaced by a second test target with an unknown presence in the sample(before testing).

FIG. 123 shows exemplary test target reagents 5758 and control targetreagents 5912 (or 5922) that may be included in system configuration5910 (or 5920) of FIG. 121 (or 122), to permit detection ofamplification signals in a different detector channel (i.e., channels 1and 2, respectively) for each nucleic acid target. Test target reagentsfor channel 1 are described above in relation to FIG. 116. Controltarget reagents 5912 (or 5922) may be similar in general structure tothe test target reagents, but different with respect to the nucleic acidsequences of the primers and probes, to provide test target and controltarget specificity, respectively. Also, the test and control probes mayinclude distinct dyes 5784, 5786 and/or distinct energy transferpartners 5798, 5930 (e.g., distinct quenchers suitable for therespective dyes). In other embodiments, at least one of the probes maybe replaced by a reporter including an intercalating dye, such as SYBRGreen.

FIGS. 124 and 125 show representative portions of exemplary data thatmay be obtained using system configuration 5910 or 5920 and the reagentsof FIG. 123. The figures show exemplary graphs 5940-5946 of fluorescencesignals that may be detected over time from a flow stream of the systemusing different detector channels, namely, a test channel (channel 1)that detects test data and a control channel (channel 2) that detectscontrol data. In FIG. 124, graph 5940 of the test data contains nopositive droplet signals. In contrast, graph 5942 of the control dataidentifies positive droplet signals, such as a positive signal 5948, ata frequency of about one in ten. Thus, the control data demonstratesthat amplification in the droplets is not inhibited substantially andsuggests that the lack of positive signals from the test data is due toan absence or undetectable level of the test target in the sample.Accordingly, the control data supports and helps to validate thenegative result in the test data. In contrast, control graph 5946 ofFIG. 125 shows no amplification of the control target (a substantiallylarger data set may be analyzed to demonstrate that the control resultholds). The control data of graph 5946 thus indicates that amplificationof the test target also is inhibited (or the sample is defective, suchas too dilute (configuration 5920)), and that the negative test resultis not valid.

FIG. 126 shows selected aspects of system of FIG. 114, with the systemin an exemplary configuration 5960 for testing amplification of a pairof nucleic acid targets in respective different (i.e., nonoverlapping)sets of droplets. Configuration 5960 may be similar to that ofconfiguration 5910, except that control reagents 5912 and controltemplate 5914 are not mixed with sample 5756 and test target reagents5758. Instead, droplets containing the control reagents and the controltemplate may be formed separately in the system, indicated at 5962, ormay be supplied as pre-formed droplets that are introduced into the flowstream downstream of droplet generator 5744, indicated at 5964.

FIG. 127 shows a pair of exemplary graphs 5980, 5982 of fluorescencesignals that may be detected over time from a flow stream of systemconfiguration 5960 of FIG. 126 using different detector channels. Graph5980 plots fluorescence signals detected from a first channel, whichdetects amplification, if any, of a test target. Graph 5982 plotsfluorescence signals detected from a second channel, which detectsamplification, if any, of a control target. Successful amplification ofthe control target, as shown here, may, for example, verify and/ormeasure aspects of the system, such as operation of the thermal cyclerand/or the detection station, the quality of the reagents, fraction ofamplification-positive droplets, or any combination thereof, amongothers.

In configuration 5960, the test and control reagents are disposedseparately in distinct droplets, so droplet signals in the first andsecond channels are not coincident, that is, they are not detected atthe same time. In other embodiments, the control target may, instead, bea second test target and the control template may, instead, be anothersample (or the same sample). Thus, the use of at least two detectorchannels permits droplets for distinct amplification tests to beinterspersed in the flow stream.

G. EXEMPLARY MULTI-CHANNEL DETECTION

FIG. 128 shows a pair of graphs 5990, 5992 illustrating exemplaryabsorption and emission spectra of fluorescent dyes that may be used inthe system of FIG. 114. The dyes are arbitrarily labeled dye 1 and dye2, respectively. However, either dye may be used to detect test signalsor control signals in the various system configurations disclosedherein. Moreover, while illustrated here for two distinguishable dyes,the system may be used for detection and analysis with three, four, ormore distinguishable dyes.

Each graph plots the intensity of absorption (“AB”), indicated at 5994,5996, and emission (“EM”), indicated at 5998, 6000, for thecorresponding dye. The dyes may have substantially overlappingabsorption spectra, such that the same wavelength of light may beutilized to excite both dyes. In contrast, the dyes may exhibit Stokesshifts (i.e., the difference (in wavelength or frequency units) betweenthe maxima of the absorption and emission spectra) of differentmagnitudes. For example, dye 1 may exhibit a smaller Stokes shift anddye 2 a larger Stokes shift, or vice versa. Accordingly, the emissionspectra of the dyes may be substantially shifted with respect to oneanother. As a result, emission from the two dyes may be detected atleast substantially independently of one another in different detectorchannels, such as a detector channel that detects light of a firstwavelength or wavelength range (e.g., λ1) and another detector channelthat detects light of a second wavelength or wavelength range (e.g.,λ2).

FIG. 129 is a schematic diagram illustrating exemplary use of thefluorescent dyes of FIG. 128 in an exemplary embodiment 6010 of system5740 of FIG. 114.

Droplets 6012 containing dyes 1 and 2, either in the same droplets ordifferent sets of droplets, may be carried in a flow stream 6014 in achannel 6016. Flow stream 6014 may pass through a detection area 6018established by an embodiment 6020 of detection station 5748.

Detection station 6020 may include a light source 6022 for exciting thefluorescent dyes in the droplets and at least one detector 6024 fordetecting light emitted from the droplets. Light source 6022 may, forexample, include an LED or laser that emits at least substantially asingle wavelength of excitation light. Alternatively, or in addition,the light source may include at least one excitation optical filter thatexcludes other wavelengths of light emanating from the light source.Detector 6024 may be equipped with detection optics 6026, 6028 (e.g.,beamsplitters, emission optical filters, separate detectors) that permitemitted light from the dyes to be detected separately.

Exemplary fluorescent dyes that may detected using system 6010 include afluorescein derivative, such as carboxyfluorescein (FAM), and a PULSAR650 dye (a derivative of Ru(bpy)₃). FAM has a relatively small Stokesshift, while Pulsar® 650 dye has a very large Stokes shift. Both FAM andPULSAR 650 dye may be excited with light of approximately 460-480 nm.FAM emits light with a maximum of about 520 nm (and not substantially at650 nm), while PULSAR 650 dye emits light with a maximum of about 650 nm(and not substantially at 520 nm). Carboxyfluorescein may be paired in aprobe with, for example, BLACK HOLE Quencher™1 dye, and PULSAR 650 dyemay be paired in a probe with, for example, BLACK HOLE Quencher™2 dye.

H. EXEMPLARY SELF-NORMALIZATION OF DROPLET SIGNALS

Test signals may be normalized using methods different from thosedescribed above in relation to FIGS. 117 and 120. In particular, themethods illustrated in FIGS. 117 and 120 involve transformation of testdata with reference data detected (a) in a different detector channel(FIG. 117) or detected (b) in different droplets (FIG. 120). Thissubsection describes methods that transform test data using aspects ofitself rather than another data set.

FIG. 130 shows a flowchart 6040 illustrating an exemplary method ofcorrecting for system fluctuations during a test. The method involvesprocessing a set of droplet test signals, shown in a first graph 6042,to produce a transformed set of test signals, shown in a second graph6044. Negative test signals 6046 and positive test signals 6048 eachshould have respective constant values over time if there is no systemvariation. However, system variation, such as the negative drift overtime illustrated in graph 6042, may produce false negatives, such as afalse negative signal 6050, and/or false positives. Transformation ofthe test signals may be performed to correct for system variation beforethe test signals are used to estimate a presence of a test target insample being tested. In particular, individual test signals may betransformed differently using the test data, accordingly to the temporalposition of each test signal. For example, each test signal may betransformed using temporally proximate test data, such as normalizationof each test signal with respect to a sliding window that averages asubset of the test signals including or adjacent the test signal. Thesubset of the test signals used may be provisionally negative, positive,or negative plus positive test signals, any of which may be re-assignedas negative/positive after transformation. For example, graph 6044 showsre-assignment of false negative signal 6050 as positive aftertransformation.

FIG. 131 shows a flowchart 6060 illustrating an exemplary method oftransforming droplet signals based on the width of respective signalpeaks providing the droplet signals. The flowchart involves graphs 6062,6064, which represent test data before and after transformation,respectively.

Graph 6062 presents test data in which the width and height of eachdroplet peak is shown. (Here, each droplet peak is presented as a squarewave to simplify the presentation. However, in other embodiments, eachdroplet peak may be detected as having any suitable shape, such as awave with sloped leading and trailing sides.) The width of a dropletfluorescence peak may be used to determine the size and volume of eachdroplet, if droplet signals are detected in a flow stream with knownflow rate, generally within a channel of fixed geometry. Knowing thevolume of sample that is tested for amplification in droplets may berequired for accurately determining the concentration/number of targetmolecules in the sample. If droplets of uniform size are desired, peakwidth may be used to identify droplets of sizes that are outside thedesired range. For example, in FIG. 131, peaks 6066, 6068 having widthsoutside a predefined range are excluded from the data set. The dropletsignals also may be transformed based on width, to provide transformedtest data (i.e., graph 6064), that has been corrected for volumevariation and/or variation in peak width.

I. SELECTED EMBODIMENTS

This subsection describes additional aspects of methods of usingcontrols and calibrations for droplet-based amplification tests, inaccordance with aspects of the present disclosure, presented withoutlimitation as a series of numbered sentences.

1. A method of sample analysis, comprising: (A) generating droplets,each droplet including first and second dyes and a partition of a sampleand being capable of amplification of a test nucleic acid target, ifpresent, in the partition; (B) detecting respective test signals andcontrol signals from the first and second dyes in the droplets, the testsignals and the control signals respectively indicating whetheramplification of the test nucleic acid target and a control nucleic acidtarget occurred in individual droplets; (C) analyzing the test signalsto determine a test result related to a presence, if any, of the testnucleic acid target in the sample; (D) analyzing the control signals todetermine a control value corresponding to a number and/or fraction ofthe droplets that are amplification-positive for the control nucleicacid target; and (E) interpreting the test result based on the controlvalue.

2. The method of paragraph 1, wherein the step of generating dropletsincludes a step of forming droplets that contain primers conferringspecificity for amplification of the control nucleic acid target.

3. The method of paragraph 2, wherein the step of forming dropletsincludes a step of forming one or more droplets that contain a controltemplate corresponding to the control nucleic acid target, and whereinthe control template is exogenous to the sample.

4. The method of paragraph 2, wherein the step of forming dropletsincludes a step of forming one or more droplets that contain a controltemplate corresponding to the control nucleic acid target, and whereinthe control template is endogenous to the sample.

5. The method of paragraph 1, wherein the step of detecting includes astep of exciting the first and second dyes with a same wavelength ofexcitation light and a step of detecting emitted light from the firstand second dyes in respective first and second detector channels.

6. The method of paragraph 1, further comprising a step of transformingthe test signals based on the control signals to reduce variation of thetest signals.

7. The method of paragraph 6, wherein the step of transforming the testsignals includes a step of transforming two or more test signalsindividually with corresponding control signals each detected from arespective same droplet as each of the two or more test signals.

8. The method of paragraph 7, wherein the step of transforming two ormore test signals includes a step of dividing each test signal by itscorresponding control signal.

9. The method of paragraph 1, wherein no more than about one in fivedroplets contain the control template.

10. The method of paragraph 1, wherein the step of analyzing the testsignals includes a step of comparing the test signals, or a transformedset of the test signals, to a signal threshold to assign individualdroplets as positive or negative for amplification of the test nucleicacid target, and a step of estimating a number of molecules of the testnucleic acid target in the sample based on results of the step ofcomparing.

11. The method of paragraph 1, wherein the step of interpreting the testresult includes a step of determining a quality of the test result.

12. The method of paragraph 11, wherein the step of determining aquality includes a step of determining the test result as being invalidif the control value is less than a threshold value.

13. A method of sample analysis, comprising: (A) generating droplets,each droplet including first and second dyes and a partition of a sampleand being capable of amplification of a test nucleic acid target, ifpresent, in the partition; (B) detecting respective test signals andreference signals from the first and second dyes in the droplets, thetest signals indicating whether amplification of the test nucleic acidtarget occurred in individual droplets; (C) transforming the testsignals based on the reference signals to reduce variation of the testsignals and to produce a set of transformed test signals; and (D)analyzing the transformed test signals to determine a test resultrelated to a presence, if any, of the test nucleic acid target in thesample.

14. The method of paragraph 13, wherein the step of transforming thetest signals includes a step of transforming two or more test signalsindividually with corresponding reference signals each detected from arespective same droplet as each of the two or more test signals.

15. The method of paragraph 14, wherein the step of transforming two ormore test signals includes a step of dividing each test signal by itscorresponding reference signal.

16. The method of paragraph 13, wherein the step of detecting includes astep of exciting the first and second dyes with a same wavelength ofexcitation light and a step of detecting emitted light from the firstand second dyes at least substantially independently from one another inrespective first and second detector channels.

17. The method of paragraph 13, wherein the step of generating dropletsincludes a step of forming droplets that contain primers conferringspecificity for amplification of a control nucleic acid target, whereinthe step of detecting includes a step of detecting control signals fromthe second dye, and wherein the control signals include the referencesignals and indicate whether amplification of the control nucleic acidtarget occurred in individual droplets.

18. The method of paragraph 17, wherein the reference signals and thecontrol signals are a same set of signals.

19. The method of paragraph 13, wherein the step of analyzing the testsignals includes a step of comparing the transformed test signals to asignal threshold to assign individual droplets as positive or negativefor amplification of the test nucleic acid target, and a step ofestimating a number of molecules of the test nucleic acid target in thesample based on results of the step of comparing.

20. A method of sample analysis, comprising: (A) generating droplets,each droplet including first and second dyes and a partition of a sampleand being capable of amplification of a test nucleic acid target, ifpresent, in the partition; (B) exciting the first and second dyes with asame wavelength of excitation light; (C) detecting emitted light fromthe first and second dyes at least substantially independently from oneanother in respective first and second detector channels to providerespective test signals and other signals measured from the first andsecond dyes in the droplets, the test signals indicating whetheramplification of the test nucleic acid target occurred in individualdroplets; and (D) analyzing the test signals to determine a test resultrelated to a presence, if any, of the test nucleic acid target in thesample, wherein the other signals are utilized to determine the testresult, to interpret the test result, to generate another test result,or any combination thereof.

21. The method of paragraph 20, wherein the other signals includereference signals, and wherein the step of analyzing includes (a) a stepof transforming the test signals based on the reference signals toreduce variation of the test signals and to produce a set of transformedtest signals and (b) a step of utilizing the set of transformed testsignals to determine the test result.

22. The method of paragraph 20, wherein the other signals includecontrol signals that indicate whether amplification of a control nucleicacid target occurred in individual droplets.

23. A method of sample analysis, comprising: (A) generating droplets,each droplet including a partition of a sample and being capable ofamplification of a nucleic acid target, if present, in the partition;(B) detecting signal peaks corresponding to the droplets, each signalpeak including a width and providing a value, the value indicatingwhether amplification of the nucleic acid target occurred in anindividual droplet; (C) transforming the value of each signal peak basedof the width of such signal peak to create a set of transformed values;(D) comparing the set of transformed values to a signal threshold to asignal threshold to assign individual droplets as positive or negativefor amplification of the test nucleic acid target; and (E) estimating apresence, if any, of the nucleic acid target in the sample based onresults of the step of comparing.

24. A method of sample analysis, comprising: (A) obtaining droplets,each droplet of at least a subset of the droplets including a partitionof a sample and being capable of amplification of a nucleic acid target,if present, in the partition; (B) detecting test signals and referencesignals from the droplets, the test signals indicating whetheramplification of the target occurred in individual droplets; (C)transforming the test signals based on the reference signals to obtaintransformed test signals; (D) comparing the transformed test signals toa signal threshold to assign individual droplets as positive or negativefor amplification of the nucleic acid target; and (E) estimating anumber of molecules of the nucleic acid target in the sample based onthe step of comparing.

25. The method of paragraph 24, wherein the step of obtaining dropletsincludes a step of obtaining test droplets and reference droplets,wherein the test droplets and the reference droplets representrespective different types of droplets, and wherein the test signals andthe reference signals are detected from the test droplets and thereference droplets, respectively.

26. The method of paragraph 25, wherein the test signals and thereference signals are detected optically at a same wavelength or samewavelength range.

27. The method of paragraph 26, wherein the test droplets and thereference droplets contain a same dye, and wherein fluorescence of thesame dye is detected as the test signals and the reference signals.

28. The method of paragraph 26, further comprising a step ofdistinguishing the test signals from the reference signals by intensity.

29. The method of paragraph 28, wherein the step of distinguishingincludes a step of interpreting, as test signals, one or more dropletsignals within a range of intensities and, as reference signals, one ormore droplet signals outside the range of intensities.

30. The method of paragraph 25, wherein the step of detecting isperformed in a detection area, and wherein the droplets travel to thedetection area in a flow stream.

31. The method of paragraph 30, wherein the test droplets and thereference droplets are intermixed in the flow stream.

32. The method of paragraph 30, wherein the test droplets are spacedfrom the reference droplets in the flow stream.

33. The method of paragraph 25, further comprising a step of thermallycycling the test droplets before the step of detecting.

34. The method of paragraph 33, wherein the reference droplets are notthermally cycled after the step of obtaining and before the step ofdetecting.

35. The method of paragraph 24, wherein the test signals and thereference signals are detected from the same droplets.

36. The method of paragraph 26, wherein the step of transformingincludes a step of transforming each test signal detected from a dropletbased on a corresponding reference signal detected from the samedroplet.

37. The method of paragraph 24, wherein the step of obtaining includes astep of generating droplets each including first and second fluorescentdyes.

38. The method of paragraph 37, wherein the step of detecting includes astep of exciting the fluorescent dyes with a same wavelength ofexcitation light and a step of detecting emission light from thefluorescent dyes at least substantially independently in respectivedetector channels.

IX. CLINICAL APPLICATIONS FOR DROPLET-BASED ASSAYS

This Section describes exemplary clinical applications for thedroplet-based assays disclosed herein. The assays may be used to performclinical (and/or forensic) tests related to etiology, pathogenesis,diagnosis, surveillance, and/or therapy monitoring of any suitableinfection, disorder, physiological condition, and/or genotype, amongothers, as illustrated below. Pathogen testing may involve pathogendetection, specification, and/or drug sensitivity applications, amongothers.

Each clinical (or non-clinical) test listed below may analyze anysuitable aspect of a particular nucleic acid target or set of two ormore targets (e.g., clinically related targets) using any suitableamplification methodology. For example, the test may be qualitative, todetermine whether or not the target (or each target) is present at adetectable, statistically significant level above background in asample, or the test may be quantitative, to determine a total presence(i.e., a concentration/copy number) of the target (or each target) inthe sample. Alternatively, or in addition, the test may determine asequence characteristic of a target (such as to determine the identityof a single nucleotide polymorphism (SNP) in the target, whether thetarget is wild-type or a variant, to genotype the target, and/or thelike). Any suitable amplification methodology may be used in performingthe tests, such as any of those described above in Section I.

The tests may provide diagnosis of a genetic disease by testing for apresence (or absence for diseases characterized by deletions) of anucleic acid target for the genetic disease. Illustrative geneticdiseases that may be diagnosed with suitable disease-specific primersinclude sickle cell anemia, cystic fibrosis (CF), Prader-Willi syndrome(PWS), beta-thalassemia, prothrombin thrombophilia, Williams syndrome,Angelman syndrome, fragile X syndrome, Factor V Leiden, or the like.Exemplary primers include hemoglobin sequences for sickle cell anemia,cystic fibrosis transmembrane conductance regulator (CFTR) genesequences for cystic fibrosis, and so on. The diagnosis may includedetermining the variant for diseases having more than one form (e.g.,distinguishing among sickle trait (AS), sickle cell anemia (SS),hemoglobin SC disease, hemoglobin SD disease, and hemoglobin SO disease,among others, for hemoglobin-related diseases). These tests may beperformed pre- or postnatally, to screen for a single disease orvariant, or for a panel of diseases and/or variants (for example, inprenatal screens, using genetic material obtained from an amniocentesisor maternal peripheral circulation, among others).

The tests may provide detection and/or delineation of native and/orpathogenic gene transcripts. For example, primers may be chosen toamplify one or more targets that signal initiation and/or amplificationof any pathophysiological messaging cascade (e.g., TNF-alpha, one ormore interleukins, NF-kappaB, one or more inflammatorymodulators/mediators), viable infectious agent proliferation, etc.

The tests may be utilized (e.g., forensically) to determine identity,paternity, maternity, sibling relationships, twin typing, genealogy,etc. These tests may be performed by amplifying nucleic acid from theindividuals at issue (including self for identity testing) and comparingnucleic acid sequences, nucleic acid restriction patterns, etc. Suitablenucleic acids may include Y-chromosome DNA for paternity testing,mitochondria DNA for maternity testing, genomic DNA for sibling tests,etc.

The tests may provide detection of viruses, their transcripts, theirdrug sensitivity, and/or pathogenic consequences thereof. For example,the tests may use primers that amplify one or more viral targets (e.g.,at least a region of one or more viral genes or transcripts), todiagnose and/or monitor viral infections, measure viral loads, genotypeand/or serotype viruses, and/or the like. Exemplary viral targets mayinclude and/or may be provided by, but are not limited to, hepatitis Cvirus (HCV), hepatitis B virus (HPB), human papilloma virus (HPV), humanimmunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein-Barr virus(EBV), respiratory syncytial virus (RSV), West Nile virus (WNV),varicella zoster virus (VZV), parvovirus, rubella virus, alphavirus,adenovirus, coxsackievirus, human T-lymphotropic virus 1 (HTLV-1),herpes virus (including for Kaposi's sarcoma), influenza virus,enterovirus, and/or the like. In some embodiments, the tests may providedetection/identification of new viral pathogens.

The tests may provide detection of prokaryotic organisms (i.e.,bacteria), their transcripts, their drug sensitivity, and/or pathogenicconsequences thereof (e.g., bacterial infections). For example, thetests may use primers that amplify one or more bacterial targets (e.g.,at least a region of one or more bacterial genes or transcripts).Suitable bacteria that may be detected include, but are not limited to,gram-positive bacteria, gram-negative bacteria, and/or other fastidiousinfectious agents. Exemplary bacterial diseases/conditions that may bediagnosed and/or monitored include sexually transmitted diseases (e.g.,gonorrhea (GC), Chlamydia (CT), syphilis, etc.); healthcare associatedinfections (HAIs), such as methicillin-resistant Staphylococcus aureus(MRSA), Clostridium difficule (C. diff.), vancomycin resistantentereococci (VRE), etc.; Group B streptococcus (GBS); mycobacteria(e.g., causing tuberculosis, leprosy, etc.); and/or the like. Furtheraspects of tests for HAIs that may be performed by the system disclosedherein are described in the following U.S. provisional patentapplications, which are incorporated herein by reference: Ser. No.61/206,975, filed Feb. 5, 2009; and Ser. No. 61/271,538, filed Jul. 21,2009.

The tests may provide detection of fungi (single-celled (e.g., yeast)and/or multi-celled), their transcripts, pathogenic consequences thereof(e.g., fungal infections), and/or drug sensitivity. For example, thetests may use primers that amplify one or more fungal targets (e.g., atleast a region of one or more viral genes or transcripts). Exemplarytypes of fungal infections that may be diagnosed and/or monitored may becaused by Histoplasma (e.g., causing histoplasmosis), Blastomyces (e.g.,causing blastomycosis), Crytococcus (e.g., causing meningitis), Coccidia(e.g., causing diarrhea), Candida, Sporothrix genuses of fungi, and/orthe like.

The tests may be used for screening, diagnosis, monitoring, and/ordesigning treatment of diseases such as cancer. For example, tests forcancer may detect one or more cancer mutations (e.g., her2/neu, BRACA-1,etc.), insertion/deletion/fusion genes (bcr-abl, k-ras, EFGR, etc.),amplified genes, epigenetic modifications, etc.; may identify cancerstem cells; may identify, monitor, and/or evaluate residual cancerdisease burden, p53 margin assessment, etc.; and/or the like. Thesetests may use any suitable cancer markers as targets and may be appliedto any suitable type of cancer, such as bladder cancer, bone cancer,breast cancer, brain cancer, cervical cancer, colorectal cancer,esophageal cancer, gastric cancer, oropharyngeal cancer, ovarian cancer,prostate cancer, uterine cancer, leukemia, lymphoma, myeloma, melanoma,etc.

The present system may be used to perform any other suitable tests. Forexample, the system may test, pre- or postnatally for aneuploidy (e.g.,Down's Syndrome, Patau Syndrome, Edwards Syndrome, Fragile X Syndrome,etc.), inborn errors of metabolism (e.g., hepatopathies,encephalopathies (e.g., acyl CoA dehydrogenase deficiency)), blood groupantigens, a congenital anomalies including but not limited tomyelomeningocele (e.g., testing for mutations inmethylenetetrahydrofolate reductase (MTHFR), methionine synthase,cystathionine beta-synthase, etc.), and/or the like. Alternatively, orin addition, the system may test for a sign of an auto-immune disorder,such as systemic lupus erythematosus (SLE), psoriasis, etc. Autoimmunetesting may include HLA classification, analysis of MHC codonbiomarkers, and/or the like. The system may test for signals ofneurodegeneration or a predisposition thereto using targets such asparkin, PINK1, tau, alpha synuclein, allele specification and tripletpenetration depth in huntingtin gene, etc. Tests may be performed forgenotyping enzymes engaged in drug metabolism (e.g., cytochrome Palleles, NAT2 polymorphisms, UGT polymorphisms, etc). Genotyping testsmay be performed in determination of susceptibility to any particulardisease state. Tests may be performed to determine clonality, such asfor immunological applications, oncology applications, etc. Tests may bedesigned to diagnose and/or monitor acute central nervous system (CNS)infection, such as encephalitis, meningitis, etc., for example, usingnucleic acid from one or more viruses and/or bacteria capable of causingthese disorders for diagnosis and nucleic acid from the virus orbacterium identified as the cause for subsequent monitoring. Tests alsomay be configured to diagnose acute ischemic disease. Suitable targetsfor this diagnosis may include CNS cellular transcripts corresponding tocirculating receptor fragments post-stroke (glutamate receptors, NMDAreceptors, 2^(nd) messenger transcripts, etc.). Other suitable targetsmay include intracellular cardiomyocyte transcripts released fromischemic cardiac regions. Tests also may be used to assess transcripts(e.g., types, numbers, etc.) in tissue proliferative disorders (e.g.,renal failure, cirrhoses, etc.). Exemplary targets for these disordersmay include trophic factors and/or extracellular matrix components,among others.

X. MULTIPLEXED ASSAYS

This Section describes exemplary strategies for performing multiplexeddetection of nucleic acid targets in the same set of droplets.

Digital PCR assays may be rendered detectable through the use of a5′-nuclease assay (i.e., a TAQMAN probe) to amplify a target sequencewhile producing a signal from the probe. The assays disclosed herein maybe configured to multiplex the 5′-nuclease assay to permit detection oftwo or more species of target from individual droplets, which is termedmultiplex detection. It is also possible to “query” for multiple targetmolecules through the presence of the target-specific reagents in eachdroplet. For example, if one were to have 50 pairs of primers eachspecific to a different gene sequence in each droplet, then each targetmolecule present will be counted for analysis. Conversely, any absenttarget molecules will not be counted. Thus, throughout this Section, theterm “query” refers to a digital “yes” or “no” determination formultiple target molecules.

It is possible to distinguish different target molecules in the samevolume based on the reporter dyes used in the 5′-nuclease assay. Thedyes differ in emission spectra so that they may be distinguished. Forexample, fluorescein, fluorescein derivatives, and rhodamine dyes may beutilized for multiplexing detection from PCR and the 5′-nuclease assay.

Different dyes could be used to code sets of 5′ nuclease reagents. Thus,one dye could signal the presence or absence of one set of 25 targetswhile a second dye could signal the presence or absence of a differentset of 25 targets. For example, one might code one set as targets fromchromosome 18 and another set from chromosome 21. One could then querychromosome 18 and 21 to count the number present based on digitalresults of two sets of assays.

It is also possible to detect different molecules in the same volumebased on the melting curve specific to homoduplexes versusheteroduplexes. A detection station may include a controllable heater toproduce one or more melting curves for each droplet, to detect multipletarget molecules by digital PCR.

Other multiplexed detection strategies may be utilized in the systemsdisclosed herein. For example, a flap endonuclease assay, commerciallyknown as an INVADER assay, is multiplexed for SNP detection in tube ormicroplate-based systems. The INVADER assay may be utilized to providedigital information in a multiplex fashion in the systems disclosedherein. The INVADER assay also can be used to query for many targetmolecules by formulating a set of two or more in every droplet. Codingis also possible.

Molecular beacon probes also may be used for multiplex detection. Theseprobes may use similar dyes to 5-nuclease assay, but the detectionmethod may be different. The structure of the probe when hybridized vs.un-hybridized produces a signal. The hybridized version produces ameasurable signal if and only if the target sequence is amplified. It ispossible to use different temperatures to multiplex detect whilesimultaneously using dyes. For example, three sets of probes may bedesigned to melt away from their target sequences at three differenttemperatures. The systems disclosed herein may produce multiplex digitalresults by both temperature and dye. Thus, in this example, the systemcan multiplex six assays with two dyes and probes that melt at threetemperatures. Also, it is possible to couple querying and coding withmolecular beacons.

An assay mixture as disclosed herein may utilize various combinations ofprimers and one or more reporters to perform a multiplexed assay.Exemplary combinations include (1) a single primer pair and notarget-specific probe (e.g., use of an intercalating dye or universalprobe as reporter for targets), (2) multiple primer pairs to amplifydistinct target species and no target-specific probe (e.g., use of anintercalating dye or universal probe), (3) multiple primer pairs toamplify distinct target species and a single color target-specific probe(e.g., a TAQMAN probe), (4) single primer pairs and multiple one colortarget-specific probes, (5) multiple primer pairs and multiple one-colortarget specific probes, or (6) multiple primer pairs and multiple colortarget specific probes, among others.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

We claim:
 1. A method of making a droplet-generating device, the methodcomprising: injection molding an upper member including a bottom surfaceand also including a first microfluidic channel, a second microfluidicchannel, and a third microfluidic channel each formed in the bottomsurface and arranged parallel thereto, the upper member having aplurality of openings each extending completely through the upper memberfrom the bottom surface, the openings creating a side wall region of asample well, a carrier well, and a droplet well; and attaching a coverlayer to the bottom surface of the upper member, such that the coverlayer seals a bottom side of each microfluidic channel; wherein themicrofluidic channels meet one another to create a droplet-generationregion, and wherein the sample well is connected to thedroplet-generation region via the first microfluidic channel, thecarrier well is connected to the droplet-generation region via thesecond microfluidic channel, and the droplet well is connected to thedroplet-generation region via the third microfluidic channel.
 2. Themethod of claim 1, wherein the upper member is injection molded toinclude a base portion that provides the bottom surface of the uppermember, and also to include a plurality of protrusions projectingupwardly from the base portion, and wherein each well has an upperportion formed by one of the protrusions.
 3. The method of claim 2,wherein the upper member is injection molded such that each protrusionis annular.
 4. The method of claim 2, wherein the cover layer is thinnerthan the base portion of the upper member.
 5. The method of claim 1,wherein the cover layer is thinner than the upper member.
 6. The methodof claim 1, wherein each opening of the plurality of openings extendsfrom a top side of the upper member to the bottom surface, and narrowsat a position intermediate the top side and the bottom surface.
 7. Themethod of claim 1, wherein the upper member is injection molded to havea fourth microfluidic channel formed in the bottom surface and arrangedparallel thereto, wherein the second microfluidic channel and the fourthmicrofluidic channel extend separately to the droplet-generation region,and wherein the carrier well also is connected to the droplet-generationregion via the fourth microfluidic channel.
 8. A method of making adroplet-generating device, the method comprising: injection molding anupper member as a single piece, the upper member having a base portionand also having a first microfluidic channel, a second microfluidicchannel, and a third microfluidic channel each formed in a bottomsurface of the base portion, the upper member including a plurality ofprotrusions projecting upwardly from the base portion, wherein one ofthe protrusions defines an upper portion of a sample well, another oneof the protrusions defines an upper portion of a carrier well, and stillanother one of the protrusions defines an upper portion of a dropletwell; and attaching a cover layer to the bottom surface of the baseportion, wherein the cover layer seals a bottom side of eachmicrofluidic channel; wherein the microfluidic channels meet one anotherin a droplet-generation region, and wherein the sample well isconfigured to supply sample-containing fluid to the droplet-generationregion via the first microfluidic channel, the carrier well isconfigured to supply carrier fluid to the droplet-generation region viathe second microfluidic channel, and the droplet well is configured toreceive sample-containing droplets and carrier fluid from thedroplet-generation region via the third microfluidic channel.
 9. Themethod of claim 8, wherein the cover layer is thinner than the baseportion.
 10. The method of claim 8, wherein the upper member defines arespective opening corresponding to each well, wherein the openingextends completely through the upper member from a top side to thebottom surface of the upper member, and wherein the upper member isinjection molded such that the opening is narrower at the bottom surfacethan the top side.
 11. The method of claim 8, wherein the upper memberis injection molded to have a fourth microfluidic channel formed in thebottom surface, wherein the second microfluidic channel and the fourthmicrofluidic channel extend separately to the droplet-generation region,and wherein the carrier well also is connected to the droplet-generationregion via the fourth microfluidic channel.